Involvement of src-kinase activation in ischemic preconditioning induced protection of mouse brain

AimsTo investigate the role of src-kinase in ischemic preconditioning induced reversal of ischemia and reperfusion induced cerebral injury in mice.Main methodsBilateral carotid artery occlusion of 17 min followed by reperfusion for 24 h was employed to produce ischemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining using both by volume and by weight methods differently. Memory was evaluated using elevated plus maze test. Rota rod test was employed to assess motor incoordination.Key findingsBilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired memory and motor co-ordination. Three preceding episodes of bilateral carotid artery occlusion for 1 min and reperfusion of 1 min (ischemic preconditioning) prevented markedly ischemia–reperfusion-induced cerebral injury measured in terms of infarct size (38.5 ± 1.3% and 38.5 ± 2.9% mean infarct of control animals was reduced to 24.3 ± 1.2% and 23.5 ± 1.8% of the preconditioning groups respectively), loss of memory (72.2 ± 3.6 mean transfer latency time of control animals was reduced to 25.6 ± 5.2 of the preconditioning group respectively) and motor coordination (78.3 ± 17.6 s mean falling down latency time of control animals was increased to a mean value of 180.9 ± 6.5 s of the preconditioning groups respectively). SU6656 (2 mg/kg, ip) and PP1 (0.1 mg/kg, ip), highly selective src-kinase inhibitors, attenuated this neuroprotective effect of ischemic preconditioning.SignificanceTherefore, neuroprotective effect of ischemic preconditioning may be due to src-kinase linked mechanism.     

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia-reperfusion injury in an in vivo rat model

AimsThis study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia–reperfusion (I/R) injury.Main methodsAnesthetized male Sprague–Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10 mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion.Key findingsMyocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo.SignificanceThese data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.     

A targeted high-efficiency angiogenesis strategy as therapy for myocardial infarction

AimsThe aim of this study was to prove that an intramyocardial injection of a mixture of low-dose human growth factor (HGF) plasmid and microbubbles (MB) in combination with insonation was an effective therapy for myocardial infarction.Main methodsTwenty dogs with myocardial infarction were divided into 4 groups: (1) HGF, MB and ultrasound (HGF-US/MB), (2) HGF and US (HGF-US), (3) HGF alone and (4) surgery alone (control). In the HGF-US/MB group, HGF plasmid DNA (500 μg) mixed with 0.5 ml of MB solution was injected 5 min after coronary occlusion followed by insonation. With the exception of the control group, the other dogs were divided into two groups, one treated with the HGF gene and insonation and the other with the HGF gene only.Key findingsCompared to the HGF group, infarct size decreased from 32% ± 7% (control) to 23% ± 5% in the HGF-US/MB group 28 d later (P < 0.05). Capillary density increased from 21.7 ± 4.2/mm² (control) to 114.3 ± 28.9/mm² in the HGF-US/MB group (P < 0.01). Compared to the HGF group, there was a 14% decrease in the ratio of left ventricle weight/body weight and a 25% decrease in hydroxyproline content. We also observed a 29% and 20% decrease in collagen volume fraction of type I and type III collagen, respectively in the HGF-US/MB group.SignificanceIntramyocardial injection of HGF and MB in combination with insonation enhances neovascularization and reduces ventricular remodeling and infarct size.     

Is preconditioning by oxytocin administration mediated by iNOS and/or mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart?

AimsOxytocin (OXT) pretreatment protects the heart during ischemia–reperfusion injury by activating ATP-dependent potassium (K_ATP) channels. The aim of the current study was to elucidate the roles of nitric oxide synthaseNOS and myocardial biochemistry in the cardioprotective effects of OXT and ischemic preconditioning (IPC).Main methodsMale New Zealand White anesthetized rabbits (13 groups) were subjected to 30 min of occlusion of the left coronary artery and 120 min of reperfusion with or without IPC.Key findingsIPC (1 cycle), OXT (0.03 μg/kg, i.p.) or IPC + OXT yield significant infarct size reductions (21.8 ± 1.5%, 20.5 ± 1.2% and 19.4 ± 1.4%, respectively, versus 38.9 ± 3.5% in the S-CONT group; P < 0.01) and antiarrhythmic effects, including VF (0%, 0% and 0%, versus 50% in S-CONT group; P < 0.05) sustained VT (13%, 13% and 13%, versus 100% in S-CONT group; P < 0.005) and other arrhythmias (25%, 13% and 25%, versus 100% in S-CONT group; P < 0.005, P < 0.01 and P < 0.005, respectively). Atosiban (ATO, a selective OXT receptor antagonist), 5-HD and l-NAME (a nonspecific NOS inhibitor) abolished the beneficial effects of IPC and OXT, suggesting that the benefits are achieved via selective activation of OXT receptors, mitochondrial K_ATP channels and NO. An iNOS inhibitor (1400 W) blocked the beneficial effects of IPC but not OXT. The IPC, OXT, IPC + OXT and 1400 W + OXT interventions significantly preserved ATP levels in the heart.SignificanceThis study demonstrates similarities between acute OXT pretreatment and IPC in terms of infarct size reduction, antiarrhythmic activity, and metabolic status.     

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AimsLate phase ischemic preconditioning (LPC) protects the heart against ischemia–reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism.Main methodsC57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC + I/R group. Myocardial tissue Po₂ and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy.Key findingsUpon reperfusion, tissue Po₂ rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mm Hg, p < 0.01). This hyperoxygenation was attenuated by LPC in the LPC + I/R mice (11.9 ± 2.0 mm Hg, p < 0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC + I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC + I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p < 0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC + I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC + I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p < 0.05).SignificanceThus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.     

Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics

AimsTo establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics.MethodsHL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA + reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca²⁺measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells.ResultsA time-dependent drop in viability was shown after HEDA (P < 0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca²⁺ peak in response to caffeine. In addition, cells subjected to HEDA for 3 h or HEDA for 3 h followed by 6 h of REPH displayed irregular Ca²⁺ oscillations with a decreased frequency.ConclusionA threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum.     

The mitochondrial Ca(2+)-activated K(+) channel contributes to cardioprotection by limb remote ischemic preconditioning in rat

AimsTo investigate the role of the mitochondrial Ca²⁺-activated K⁺ channel in cardioprotection induced by limb remote ischemic preconditioning.Main methodsMale Sprague–Dawley rats (250–300 g) were randomized into control, ischemia/reperfusion (I/R), remote ischemic preconditioning (RPC), NS1619 (a specific mitochondrial Ca²⁺-activated K⁺ channel opener), and RPC + paxilline (a specific mitochondrial Ca²⁺-activated K⁺ channel inhibitor) groups. RPC was induced by 4 cycles of 5 min of ligation followed by 5 min of reperfusion of the left femoral artery. Myocardial I/R was achieved by ligation of the left anterior descending coronary artery for 30 min, followed by 120 min of reperfusion. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, the hemodynamics were monitored, and lactate dehydrogenase (LDH) levels in the coronary effluent, manganese superoxide dismutase (Mn-SOD) content in mitochondria and mitochondrial membrane potential were measured spectrophotometrically. The ultrastructure of cardiomyocyte mitochondria was assessed by electron microscopy.Key findingsNS1619 (10 μM) improved heart function, decreased infarct size, reduced LDH release, maintained mitochondrial structural integrity and mitochondrial membrane potential, and increased the mitochondrial content of Mn-SOD to the same degree as RPC treatment. However, paxilline (1 μM) eliminated the cardioprotective effect conferred by RPC.SignificanceThe mitochondrial Ca²⁺-activated K⁺ channel participates in the myocardial protection by limb remote ischemic preconditioning.     

Methane output of rabbits (Oryctolagus cuniculus) and guinea pigs (Cavia porcellus) fed a hay-only diet: implications for the scaling of methane production with body mass in non-ruminant mammalian herbivores

It is assumed that small herbivores produce negligible amounts of methane, but it is unclear whether this is a physiological peculiarity or simply a scaling effect. A respiratory chamber experiment was conducted with six rabbits (Oryctolagus cuniculus, 1.57 ± 0.31 kg body mass) and six guinea pigs (Cavia porcellus, 0.79 ± 0.07 kg) offered grass hay ad libitum. Daily dry matter (DM) intake and DM digestibility were 50 ± 6 g kg^? 0.75 d^? 1 and 55 ± 6% in rabbits and 59 ± 11 g kg^? 0.75 d^? 1 and 61 ± 3% in guinea pigs, respectively. Methane production was similar for both species (0.20 ± 0.10 L d^? 1 and 0.22 ± 0.08 L d^? 1) and represented 0.69 ± 0.32 and 1.03 ± 0.29% of gross energy intake in rabbits and guinea pigs, respectively. In relation to body mass (BM) guinea pigs produced significantly more methane. The data on methane per unit of BM obtained in this study and from the literature on the methane output of elephant, wallabies and hyraxes all lay close to a regression line derived from roughage-fed horses, showing an increase in methane output with BM. The regression, including all data, was nearly identical to that based on the horse data only (methane production in horses [L d^? 1] = 0.18 BM [kg]^{0.97 (95%CI 0.92–1.02)}) and indicates linear scaling. Because feed intake typically scales to BM^0.75, linear scaling of methane output translates into increasing energetic losses at increasing BM. Accordingly, the data collection indicates that an increasing proportion of ingested gross energy is lost because relative methane production increases with BM. Different from ruminants, such losses (1%–2% of gross energy) appear too small in non-ruminant herbivores to represent a physiologic constraint on body size. Nevertheless, this relationship may represent a physiological disadvantage with increasing herbivore body size.     

Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4 gene transfer create cardiac pacemakers

Background aimsThe study objective was to test the ability of canine mesenchymal stromal cells (cMSC) transfected with the mouse hyperpolarization-activated cyclic nucleotide-gated channel 4 (mHCN4) gene to deliver a biologic pacemaker to the canine heart.Methods and ResultscMSC that were transfected by lentiviral vector with the cardiac pacemaker gene mHCN4 expressed high levels of Cs⁺ -sensitive current (26.4 ± 1.8pA/pF at –140 mV; (n = 17) and were activated in the diastolic potential range with a reversal potential of –29.7 ± 2.5 mV (n = 14), confirming that the expressed current was Funny current (I_f)-like. Next, 3 × 10⁶ cMSC transfected with either control plasmid or the mHCN4 gene construct were injected subepicardially into the canine right ventricular wall in situ. During sinus arrest, all control hearts had spontaneous atrioventricular node rhythms [rate = 21 ± 5beats per minute (b.p.m.)]. In the mHCN4 group, six of eight animals developed spontaneous ventricular rhythms of right-sided origin (rate = 45 ± 9b.p.m.; P < 0.01). Moreover, immunohistochemical analysis of the injected regions demonstrated neither apoptosis nor cellular or humoral rejection at 2 weeks.ConclusionsThese results demonstrate that genetically modified cMSC can express functional HCN4 channels in vitro and in vivo and represent a novel delivery system for pacemaker genes into the heart.     

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Background aimsDonor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.MethodsWe performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler.ResultsThe majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10¹⁰ total cells, 6.2 ± 2.2 × 10⁸ of which were CD45⁺ CD34⁺. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34⁺ CD90⁺ CD117^dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 ⁺ cells) and CD45^? CD73⁺ CD105⁺ mesenchymal stromal cells (0.04 ± 0.04% of the total cells).ConclusionsThis procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.     

CpG-ODN,the TLR9 agonist,attenuates myocardial ischemia/reperfusion injury: Involving activation of PI3K/Akt signaling

BackgroundToll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury.MethodsMale C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1 h prior to myocardial ischemia (60 min) followed by reperfusion. Untreated mice served as I/R control (n = 10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14 days.ResultsCpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14 days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection.ConclusionCpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.     

Periosteal microcirculatory action of chronic estrogen supplementation in osteoporotic rats challenged with tourniquet ischemia

AimsTransient ischemia of osteoporotic bones during elective orthopedic surgery or fracture repair carries risks for serious complications, and estrogen loss or replacement has a potential to influence ischemia–reperfusion-induced inflammatory activation. To clarify this, we investigated the periosteal inflammatory changes in a clinically relevant time frame in ovariectomized rats, an experimental model of postmenopausal bone loss. Furthermore, the effects of chronic estrogen supplementation on the postischemic local and systemic inflammatory reactions were assessed.Main methodsBilateral ovariectomy or sham operation was performed in 3-month-old female Sprague–Dawley rats. Five months later, estrogen replacement therapy with 17β-estradiol (20 μg^? 1 kg^? 1 day^? 1) or vehicle treatment was initiated. The microcirculatory inflammatory consequences of 60-min total hindlimb ischemia followed by 180-min reperfusion were examined 11 months after ovariectomy and were compared with those in 3-month-old animals.Key findingsThe osteoporosis that developed 5 months after ovariectomy was significantly ameliorated by estrogen replacement therapy. Both in ovariectomized and in non-ovariectomized animals, ischemia–reperfusion elevated the neutrophil adherence ~ 3-fold in the postcapillary venules of the periosteum (intravital microscopy), with an ~ 50–60% increase in intravascular neutrophil activation (CD11b; FACS analysis), an enhanced TNF-α release (ELISA) and periosteal expression of ICAM-1 (the endothelial ligand of CD11b; immunohistochemistry). Exogenous 17β-estradiol considerably reduced TNF-α release and the number of neutrophil–endothelial interactions in the periosteum, without affecting the CD11b and ICAM-1 expression changes.SignificanceOsteoporosis itself does not increase the magnitude of the limb ischemia–reperfusion-associated periosteal inflammatory reaction. Chronic estrogen supplementation, however, reverses osteoporosis and significantly ameliorates the microcirculatory consequences of transient ischemia.     

Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy

Background and AimsWith the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34⁺ selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices.Methods and ResultsWe developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10⁶ CD34⁺ cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34⁺ bone marrow cells revealed a significant proportion of CD45^? CD34⁺ cells.ConclusionsIntra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45^? cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.     

Évaluation de la sidération myocardique post-effort immédiat par tomoscintigraphie myocardique au 99mTc-sestamibi couplée à l’ECG

AimRepeated episodes of myocardial stunning may lead to chronic ventricular dysfunction. We attempted to assess the parameters related to post-exercise stunning in patients undergoing gated SPECT.MethodsSix hundred patients undergoing a one-day stress/rest ^99mTc-sestamibi gated SPECT were studied. Stress imaging was acquired within 15 minutes after injection. Summed perfusion scores (SSS, SRS and SDS) were calculated using QPS, and LV function assessed using QGS. Stunning was defined as the association of ischemia (SSS ≥ 4 and SDS > 0) and a minimum of 5% decrease in post-stress EF.ResultsIschemia was found in 225 (37.5%) patients. Among these, 67 (30%) showed myocardial stunning. Patients with stunning had a lower rest ESV (47 ± 24 mL vs 65 ± 52 mL, p < 0.0003) and EDV (108 ± 35 mL vs 122 ± 66 mL, p = 0.03), an increased rest LVEF (58 ± 10% vs 52 ± 13%, p < 0.0001) and a decreased post-stress LVEF (49 ± 10% vs 53 ± 13%, p < 0.02) compared to patients with no stunning. The number of myocardial segments showing reversible perfusion defects was increased in patients with stunning (2.7 ± 2.6 vs 1.7 ± 2.1, p < 0.02). On logistic regression, an extent of ischemia greater than two segments and a rest EF greater than 45% were independent predictors of the occurrence of myocardial stunning in patients with ischemia.ConclusionsIn patients with ischemia, exercise-induced stunning was associated with an increased extent of ischemia but also preserved rest ventricular function.     

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

Background aimsThe ability of hematopoietic progenitor cells–apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented.MethodsIn this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2–7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage).ResultsThe doses of nucleated and CD34⁺ cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10⁸ and 6.8 ± 4.3 × 10⁶, respectively) and long- (4.0 ± 4.9 × 10⁸ and 6.1 ± 3.4 × 10⁶, respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10⁹/L (median 11 days for both short- and long-term storage) and platelets >20 × 10⁹/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10⁶/kg CD34⁺ cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets).ConclusionsCryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.     

Novel l-adenosine analogs as cardioprotective agents

Two l-nucleosides, l-3′-amino-3′-deoxy-N⁶-dimethyladenosine (l-3′-ADMdA) 1, previously synthesized in our laboratory, and the novel l-3′-amino-3′-deoxy-N⁶-methyladenosine-5′-N-methyluronamide (l-3′-AM-MECA) 2 were evaluated in an ischemia/reperfusion model on Langendorff perfused mouse heart. l-3′-ADMdA 1 was found to enhance functional recovery from ischemia (32.2 ± 3.7 cm H₂O/s % rate pressure product, compared to 21.3 ± 1.4 for the control and 30.7 ± 3.4 for adenosine) and increase the time to onset of ischemic contracture (14.5 ± 0.9 min, compared to 10.5 ± 1.0 min for the control and 13.6 ± 0.6 min for adenosine) comparable to adenosine. Consistent with the functional recovery data, decreased infarction area was seen in the case of 1 (19.1 ± 8.4, compared to 40.5 ± 7.2% for the control and 11.5 ± 2.1% for adenosine). In contrast, l-3′-AM-MECA 2 did not show significant functional recovery, increased onset of contracture, nor decreased infarction area compared to control. Unlike adenosine, neither 1 nor 2 induced cardiac standstill in mouse heart.     

Transmyocardial drilling revascularization combined with heparinized bFGF-incorporating stent activates resident cardiac stem cells via SDF-1/CXCR4 axis

ObjectiveTo investigate whether transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor (bFGF)-incorporating degradable stent implantation (TMDRSI) can promote myocardial regeneration after acute myocardial infarction (AMI).MethodsA model of AMI was generated by ligating the mid-third of left anterior descending artery (LAD) of miniswine. After 6 h, the animals were divided into none-treatment (control) group (n = 6) and TMDRSI group (n = 6). For TMDRSI group, two channels with 3.5 mm in diameter were established by a self-made drill in the AMI region, into which a stent was implanted. Expression of stromal cell-derived factor-1_α (SDF-1_α) and CXC chemokine receptor 4 (CXCR4), cardiac stem cell (CSC)-mediated myocardial regeneration, myocardial apoptosis, myocardial viability, and cardiac function were assessed at various time-points.ResultsSix weeks after the operation, CSCs were found to have differentiated into cardiomyocytes to repair the infarcted myocardium, and all above indices showed much improvement in the TMDRSI group compared with the control group (P < 0.001).ConclusionsThe new method has shown to be capable of promoting CSCs proliferation and differentiation into cardiomyocytes through activating the SDF-1/CXCR4 axis, while inhibiting myocardial apoptosis, thereby enhancing myocardial regeneration following AMI and improving cardiac function. This may provide a new strategy for myocardial regeneration following AMI.     

Adenovirus-mediated tissue factor pathway inhibitor gene transfer induces apoptosis by blocking the phosphorylation of JAK-2/STAT-3 pathway in vascular smooth muscle cells

ObjectiveIn our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated.MethodsRat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis.ResultsTFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91 ± 1.66%, 13.46 ± 1.28% and 17.04 ± 1.95%, respectively, whereas those in the LacZ group were 3.28 ± 0.89%, 4.01 ± 0.72% and 4.89 ± 1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P < 0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P > 0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd, 5th and 7th days after gene transfer.ConclusionThe results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis.