Targeted muscle reinnervation prevents and reverses rat pain behaviors after nerve transection.

ABSTRACT: Targeted muscle reinnervation (TMR) is a clinical intervention that is rapidly becoming common in major limb amputation to prevent or reduce amputation-related pain. However, TMR is much less effective when applied long after injury compared with acute TMR. Since the mechanisms governing pain relief in TMR of amputated nerves are unknown, we developed a preclinical model as a platform for mechanistic examination. Following spared nerve injury (SNI), rats underwent either TMR, simple neuroma excision, or a sham manipulation of the injury site. These interventions were performed immediately or delayed (3 or 12 weeks) after SNI. Pain behavior was measured as sensitivity to mechanical stimuli (pin, von Frey, and dynamic brush) and thermal stimuli (acetone and radiant heat). Spared nerve injury produced hypersensitivity to all mechanical stimuli and cold, which persisted after sham surgery. Targeted muscle reinnervation at the time of SNI prevented the development of pain behaviors and performing TMR 3 weeks after SNI reversed pain behaviors to baseline. By contrast, TMR performed at 12 weeks after SNI had no effect on pain behaviors. Neuroma excision resulted in significantly less reduction in hyperalgesia compared with TMR when performed 3 weeks after SNI but had no effect at 12 weeks after SNI. In this model, the pain phenotype induced by nerve transection is reduced by TMR when performed within 3 weeks after injury. However, TMR delayed 12 weeks after injury fails to reduce pain behaviors. This replicates clinical experience with limb amputation, supporting validity of this model for examining the mechanisms of TMR analgesia.

Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa).

INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.

Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation.

Tissue damage and necrosis from inflammatory processes are a consequence of ischemia reperfusion injury (IRI). In skeletal muscle, ischemia reduces the aerobic energy capacity of muscle cells, leading to adverse biochemical alterations and inflammation. The goal of this study is to show that exposure to near-infrared light (NIR) during a period of ischemia reduces IRI by decreasing necrosis and inflammation in addition to decreasing proinflammatory M1 and increasing protective M2 macrophages. C57/Bl6 mice underwent unilateral tourniquet-induced hindlimb ischemia for 3 h followed by reperfusion for either 15 or 30 min. Mice were randomly assigned to 3 groups. Group 1 underwent IRI with 30 min reperfusion. Group 2 underwent IRI with a 15 min reperfusion. Each group consisted of 50% no-NIR and 50% NIR-treated mice with exposure of 50 mW/cm2 for 5 min/1 h after tourniquet closure. Group 3 were sham animals anesthetized for 3 h omitting IRI. Laser doppler flow imaging was performed on all mice to confirm ischemia and reperfusion. Flow data were expressed as the ratio of ischemic limb and the contralateral control. The mice were euthanized after reperfusion, and the quadriceps and gastrocnemius were harvested. Immunoprecipitation and western blot of macrophage-markers CD68 (M1) and CD206 (M2) were performed and normalized to CD14 expression. The expression of the inflammatory markers CXCL1 and CXCL5 was significantly reduced by NIR in the IRI group. A significant decrease in CD68 and an increase in CD206 expression was observed in animals receiving IR and NIR. Tissue necrosis was decreased by NIR in the IRI group, as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The findings demonstrate that exposure to NIR reduced IRI and improved tissue survival. NIR reduced inflammation, decreased proinflammatory M1, and increased protective M2 macrophages. Exposure to NIR reduced inflammation and enhanced regeneration, leading to tissue protection following ischemia.

Adjuvant doxycycline to enhance anti-amyloid effects: Results from the dual phase 2 trial.

BACKGROUND: Although, doxycycline use is associated with improved outcomes in amyloidosis in retrospective studies, evidence from clinical trials is limited. METHODS: This phase 2 trial of doxycycline (clinicaltrials.gov: NCT02207556) in newly diagnosed light chain (AL) amyloidosis enrolled 25 patients with systemic AL amyloidosis on treatment with doxycycline for 1 year along with chemotherapy. Outcomes of interest included mortality, organ response, and hematologic response rates at 1 year. FINDINGS: The median age was 62 years, 64% were male, and 68% had the AL lambda subtype. Patients had Mayo 2012 stage 3 in 24% and stage 4 in 28%. Cardiac involvement was present in 60% of patients, renal involvement in 72%, and 60% patients had 3 or more organs involved. Target organ was cardiac in 14(56%), renal in 7(28%), hepatic in 1(4%) and soft tissue in 3(12%). At 1 year, mortality was 20% (95% confidence interval, 8.9-41.6%) and organ response was 36% (18-57%). Hematologic response in 1-year survivors was 100%, including 30% complete and 55% very good partial response. Autologous hematopoietic cell transplant was performed in 60%; among transplanted patients, day-100 transplant-related mortality was 0. Doxycycline use was safe and not attributed to any grade 2 or higher toxicity. INTERPRETATION: In addition to a low 1-year mortality, doxycycline use was safe and associated with high transplant utilization rate. We thus contend that doxycycline should be studied in a placebo-controlled study in newly diagnosed AL patients in the first year, particularly among patients with advanced disease and cardiac involvement.

Inhibition of myeloperoxidase increases revascularization and improves blood flow in a diabetic mouse model of hindlimb ischaemia.

OBJECTIVE: Diabetes mellitus is a significant risk factor for peripheral artery disease. Diabetes mellitus induces chronic states of oxidative stress and vascular inflammation that increase neutrophil activation and release of myeloperoxidase. The goal of this study is to determine whether inhibiting myeloperoxidase reduces oxidative stress and neutrophil infiltration, increases vascularization, and improves blood flow in a diabetic murine model of hindlimb ischaemia. METHODS: Leptin receptor-deficient (db/db) mice were subjected to hindlimb ischaemia. Ischaemic mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC) to inhibit myeloperoxidase. After ligating the femoral artery, effects of treatments were determined with respect to hindlimb blood flow, neutrophil infiltration, oxidative damage, and the capability of hindlimb extracellular matrix to support human endothelial cell proliferation and migration. RESULTS: KYC treatment improved hindlimb blood flow at 7 and 14 days in db/db mice; decreased the formation of advanced glycation end products, 4-hydroxynonenal, and 3-chlorotyrosine; reduced neutrophil infiltration into the hindlimbs; and improved the ability of hindlimb extracellular matrix from db/db mice to support endothelial cell proliferation and migration. CONCLUSION: These results demonstrate that inhibiting myeloperoxidase reduces oxidative stress in ischaemic hindlimbs of db/db mice, which improves blood flow and reduces neutrophil infiltration such that hindlimb extracellular matrix from db/db mice supports endothelial cell proliferation and migration.

Wavelength-dependence of vasodilation and NO release from S-nitrosothiols and dinitrosyl iron complexes by far red/near infrared light.

Far red/near infrared (R/NIR) energy is a novel therapy, but its mechanism of action is poorly characterized. Cytochrome c oxidase (Cco) of the mitochondrial electron transport chain is considered the primary photoacceptor for R/NIR to photolyze a putative heme nitrosyl in Cco to liberate free nitric oxide (NO). We previously observed R/NIR light directly liberates NO from nitrosylated hemoglobin and myoglobin, and recently suggested S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNIC) may be primary sources of R/NIR-mediated NO. Here we indicate R/NIR light exposure induces wavelength dependent dilation of murine facial artery, with longer wavelengths (740, and 830 nm) exhibiting reduced potency when compared to 670 nm. R/NIR also stimulated NO release from pure solutions of low molecular weight RSNO (GSNO and SNAP) and glutathione dinitrosyl iron complex (GSH-DNIC) in a power- and wavelength-dependent manner, with the greatest effect at 670 nm. NO release from SNAP using 670 was nearly ten-fold more than GSNO or GSH-DNIC, with no substantial difference in NO production at 740 nm and 830 nm. Thermal effects of irradiation on vasodilation or NO release from S-nitrosothiols and DNIC was minimal. Our results suggest 670 nm is the optimal wavelength for R/NIR treatment of certain vascular-related diseases.

Rationale and design of DUAL study: Doxycycline to Upgrade response in light chain (AL) amyloidosis (DUAL): A phase 2 pilot study of a two-pronged approach of prolonged doxycycline with plasma cell-directed therapy in the treatment of AL amyloidosis.

Light chain (AL) amyloidosis is a plasma cell neoplasm associated with insoluble fibril deposition from clonal immunoglobulin chains systemically. The disease is associated with high early mortality and morbidity owing to advanced organ deposition as well as lack of proven de-fibrillogenic therapies. Pre-clinical and retrospective clinical data suggests that doxycycline has benefit in AL amyloidosis. The ongoing DUAL study is a single center, open label, phase 2 study in which patients with AL amyloidosis who are undergoing clone-directed therapy for the underlying neoplasm with oral doxycycline given for 1 year to test the hypothesis that prolonged doxycycline use will be safe, feasible, and lead to reduced early mortality in systemic AL amyloidosis and hasten organ amyloid response. Clinical follow up visits will occur at monthly intervals for systemic AL patients and at 3 monthly intervals for localized AL patients. Blood tests will be collected during these time points for hematologic response assessment. Organ testing will be conducted at 3 monthly intervals and radiologic testing will be conducted at 6 monthly intervals. Research blood samples will be collected at baseline, 6 and 12 months. Other correlative studies include matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP) testing and patient-reported outcomes.

An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice.

Interferon regulatory factor 5 (IRF5) has been called a "master switch" for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0-100 µg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50µg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.

N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor.

Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (≤4,000 µM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and human promyelocytic leukemia (HL-60) cells but not superoxide generation by PMA-stimulated HL-60 cells. KYC inhibits MPO-mediated HOCl formation in BAEC culture and protects BAECs from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. Ultraviolet-visible spectral studies indicate KYC binds to the active site of MPO and reacts with compounds I and II. Docking studies show the Tyr of KYC rests just above the heme of MPO. Interestingly, KYC increases MPO-dependent H2O2 consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures. Accordingly, KYC may be useful for treating MPO-mediated vascular disease.

Far red/near infrared light treatment promotes femoral artery collateralization in the ischemic hindlimb.

Nitric oxide (NO) is a crucial mediator of hindlimb collateralization and angiogenesis. Within tissues there are nitrosyl-heme proteins which have the potential to generate NO under conditions of hypoxia or low pH. Low level irradiation of blood and muscle with light in the far red/near infrared spectrum (670 nm, R/NIR) facilitates NO release. Therefore, we assessed the impact of red light exposure on the stimulation of femoral artery collateralization. Rabbits and mice underwent unilateral resection of the femoral artery and chronic R/NIR treatment. The direct NO scavenger carboxy-PTIO and the nitric oxide synthase (NOS) inhibitor L-NAME were also administered in the presence of R/NIR. DAF fluorescence assessed R/NIR changes in NO levels within endothelial cells. In vitro measures of R/NIR induced angiogenesis were assessed by endothelial cell proliferation and migration. R/NIR significantly increased collateral vessel number which could not be attenuated with L-NAME. R/NIR induced collateralization was abolished with c-PTIO. In vitro, NO production increased in endothelial cells with R/NIR exposure, and this finding was independent of NOS inhibition. Similarly R/NIR induced proliferation and tube formation in a NO dependent manner. Finally, nitrite supplementation accelerated R/NIR collateralization in wild type C57Bl/6 mice. In an eNOS deficient transgenic mouse model, R/NIR restores collateral development. In conclusion, R/NIR increases NO levels independent of NOS activity, and leads to the observed enhancement of hindlimb collateralization.

Intraganglionic AAV6 results in efficient and long-term gene transfer to peripheral sensory nervous system in adult rats.

We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10(9) viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.

Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse.

The tight skin mouse (Tsk(-/+)) is a model of scleroderma characterized by impaired vasoreactivity, increased oxidative stress, attenuated angiogenic response to VEGF and production of the angiogenesis inhibitor angiostatin. Low-level light therapy (LLLT) stimulates angiogenesis in myocardial infarction and chemotherapy-induced mucositis. We hypothesize that repetitive LLLT restores vessel growth in the ischemic hindlimb of Tsk(-/+) mice by attenuating angiostatin and enhancing angiomotin effects in vivo. C57Bl/6J and Tsk(-/+) mice underwent ligation of the femoral artery. Relative blood flow to the foot was measured using a laser Doppler imager. Tsk(-/+) mice received LLLT (670 nm, 50 mW cm(-2), 30 J cm(-2)) for 10 min per day for 14 days. Vascular density was determined using lycopersicom lectin staining. Immunofluorescent labeling, Western blot analysis and immunoprecipitation were used to determine angiostatin and angiomotin expression. Recovery of blood flow to the ischemic limb was reduced in Tsk(-/+) compared with C57Bl/6 mice 2 weeks after surgery. LLLT treatment of Tsk(-/+) mice restored blood flow to levels observed in C57Bl/6 mice. Vascular density was decreased, angiostatin expression was enhanced and angiomotin depressed in the ischemic hindlimb of Tsk(-/+) mice. LLLT treatment reversed these abnormalities. LLLT stimulates angiogenesis by increasing angiomotin and decreasing angiostatin expression in the ischemic hindlimb of Tsk(-/+) mice.

4F decreases IRF5 expression and activation in hearts of tight skin mice.

The apoAI mimetic 4F was designed to inhibit atherosclerosis by improving HDL. We reported that treating tight skin (Tsk(-/+)) mice, a model of systemic sclerosis (SSc), with 4F decreases inflammation and restores angiogenic potential in Tsk(-/+) hearts. Interferon regulating factor 5 (IRF5) is important in autoimmunity and apoptosis in immune cells. However, no studies were performed investigating IRF5 in myocardium. We hypothesize that 4F differentially modulates IRF5 expression and activation in Tsk(-/+) hearts. Posterior wall thickness was significantly increased in Tsk(-/+) compared to C57Bl/6J (control) and Tsk(-/+) mice with 4F treatment assessed by echoradiography highlighting reduction of fibrosis in 4F treated Tsk(-/+) mice. IRF5 in heart lysates from control and Tsk/+ with and without 4F treatment (sc, 1 mg/kg/d, 6-8 weeks) was determined. Phosphoserine, ubiquitin, ubiquitin K(63) on IRF5 were determined on immunoprecipitates of IRF5. Immunofluorescence and TUNEL assays in heart sections were used to determine positive nuclei for IRF5 and apoptosis, respectively. Fluorescence-labeled streptavidin (SA) was used to determine endothelial cell uptake of biotinylated 4F. SA-agarose pulldown and immunoblotting for IRF5 were used to determine 4F binding IRF5 in endothelial cell cytosolic fractions and to confirm biolayer interferometry studies. IRF5 levels in Tsk(-/+) hearts were similar to control. 4F treatments decrease IRF5 in Tsk(-/+) hearts and decrease phosphoserine and ubiquitin K(63) but increase total ubiquitin on IRF5 in Tsk(-/+) compared with levels on IRF5 in control hearts. 4F binds IRF5 by mechanisms favoring association over dissociation strong enough to pull down IRF5 from a mixture of endothelial cell cytosolic proteins. IRF5 positive nuclei and apoptotic cells in Tsk(-/+) hearts were increased compared with controls. 4F treatments decreased both measurements in Tsk(-/+) hearts. IRF5 activation in Tsk(-/+) hearts is increased. 4F treatments decrease IRF5 expression and activation in Tsk(-/+) hearts by a mechanism related to 4F's ability to bind IRF5.

Myocardial interstitial fluid inhibits proliferation and cardiomyocyte differentiation in pluripotent embryonic stem cells.

Several recent studies have demonstrated that the transplantation of pluripotent murine embryonic stem cells (mESCs) can improve or restore the function of infarcted myocardium. Although the extent of remuscularization and its contribution to the restoration of function are unclear, these outcomes are likely strongly influenced by factors in the infarcted and/or ischemic environment. As an initial step toward understanding how the ischemic environment of host myocardium affects transplanted pluripotent cells, we have taken a reductionist approach wherein mESCs are cultured in medium containing ischemic myocardial interstitial fluid (iMIF). iMIF is generated in canine myocardium during eight hourly episodes of transient ischemia and collected on a daily basis, over a 24-day collection period. iMIF strongly reduced the numbers of pluripotent mESCs after 11 days in culture. This inhibitory effect, which was most pronounced for iMIF pools from early time points of the 24-day collection period, resulted from an inhibition of cell proliferation. iMIF also inhibited the differentiation of pluripotent mESCs into cardiomyocytes. By contrast, the expression of vascular smooth muscle and endothelial cell markers was relatively unaffected, consistent with previous findings that iMIF promotes angiogenesis. Taken together, these results suggest that whereas the ischemic/infarcted environment is favorable to stem cell-mediated angiogenesis, it is hostile to cardiac myogenesis. These findings also imply that observations of mESC-mediated improvement of cardiac function after transplantation of pluripotent cells do not reflect remuscularization.

Mechanism of differential cardiovascular response to propofol in Dahl salt-sensitive, Brown Norway, and chromosome 13-substituted consomic rat strains: role of large conductance Ca2+ and voltage-activated potassium channels.

Cardiovascular sensitivity to general anesthetics is highly variable among individuals in both human and animal models, but little is known about the genetic determinants of drug response to anesthetics. Recently, we reported that propofol (2,6-diisopropylphenol) causes circulatory instability in Dahl salt-sensitive SS/JRHsdMcwi (SS) rats but not in Brown Norway BN/NHsdMcwi (BN) rats and that these effects are related to genes on chromosome 13. Based on the hypothesis that propofol does target mesenteric circulation, we investigated propofol modulation of mesenteric arterial smooth muscle cells (MASMC) in SS and BN rats. The role of chromosome 13 was tested using SS-13(BN)/Mcwi and BN-13(SS)/Mcwi consomic strains with chromosome 13 substitution. Propofol (5 microM) produced a greater in situ hyperpolarization of MASMC membrane potential in SS than BN rats, and this effect was abrogated by iberiotoxin, a voltage-activated potassium (BK) channel blocker. In inside-out patches, the BK channel number, P(o), and apparent Ca(2+) sensitivity, and propofol sensitivity all were significantly greater in MASMC of SS rats. The density of whole-cell BK current was increased by propofol more in SS than BN myocytes. Immunolabeling confirmed higher expression of BK alpha subunit in MASMC of SS rats. Furthermore, the hyperpolarization produced by propofol, the BK channel properties, and propofol sensitivity were modified in MASMC of SS-13(BN)/Mcwi and BN-13(SS)/Mcwi strains toward the values observed in the background SS and BN strains. We conclude that differential function and expression of BK channels, resulting from genetic variation within chromosome 13, contribute to the enhanced propofol sensitivity in SS and BN-13(SS)/Mcwi versus BN and SS-13(BN)/Mcwi strains.

Inhibition of glycogen synthase kinase or the apoptotic protein p53 lowers the threshold of helium cardioprotection in vivo: the role of mitochondrial permeability transition.

BACKGROUND: Prosurvival signaling kinases inhibit glycogen synthase kinase-3beta (GSK-3beta) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS: Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-alpha (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS: Helium reduced (P < 0.05) myocardial infarct size (35 +/- 6 [n = 7], 25 +/- 4 [n = 7], and 20 +/- 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 +/- 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS: Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo.

Reactive oxygen species and mitochondrial adenosine triphosphate-regulated potassium channels mediate helium-induced preconditioning against myocardial infarction in vivo.

OBJECTIVES: Helium produces preconditioning by activating prosurvival kinases, but the roles of reactive oxygen species (ROS) or mitochondrial adenosine triphosphate-regulated potassium (K(ATP)) channels in this process are unknown. The authors tested the hypothesis that ROS and mitochondrial K(ATP) channels mediate helium-induced preconditioning in vivo. DESIGN: A randomized, prospective study. SETTING: A university research laboratory. PARTICIPANTS: Male New Zealand white rabbits. INTERVENTIONS: Rabbits (n = 64) were instrumented for the measurement of systemic hemodynamics and subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. In separate experimental groups, rabbits (n = 7 or 8 per group) were randomly assigned to receive 0.9% saline (control) or 3 cycles of 70% helium-30% oxygen administered for 5 minutes interspersed with 5 minutes of an air-oxygen mixture before LAD occlusion with or without the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2 mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the mitochondrial K(ATP) antagonist 5-hydroxydecanoate (5-HD; 5 mg/kg). Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni's modification of a Student t test. MEASUREMENTS AND MAIN RESULTS: The myocardial infarct size was determined by using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium significantly (p < 0.05) reduced infarct size (23 +/- 4% of the area at risk; mean +/- standard deviation) compared with control (46 +/- 3%). NAC, 2-MPG, and 5-HD did not affect irreversible ischemic injury when administered alone (49 +/- 5%, 45 +/- 6%, and 45 +/- 3%), but these drugs blocked reductions in infarct size produced by helium (45 +/- 4%, 45 +/- 2%, and 44 +/- 3%). CONCLUSIONS: The results suggest that ROS and mitochondrial K(ATP) channels mediate helium-induced preconditioning in vivo.

Inhibition of apoptotic protein p53 lowers the threshold of isoflurane-induced cardioprotection during early reperfusion in rabbits.

INTRODUCTION: Exposure to isoflurane before and during early reperfusion protects against myocardial infarction by activating phosphatidylinositol-3-kinase (PI3K)-mediated signaling. The apoptotic protein, p53, is regulated by PI3K, and inhibition of p53 protects against ischemic injury. We tested the hypothesis that p53 inhibition lowers the threshold of isoflurane-induced postconditioning in vivo. METHODS: Rabbits (n = 73) instrumented for hemodynamic measurement and subjected to a 30-min left anterior descending coronary artery occlusion and 3-h reperfusion received 0.9% saline (control), isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) administered for 3 min before and 2 min after reperfusion, the p53 inhibitor pifithrin-alpha (1.5 or 3.0 mg/kg), or 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha. Other rabbits received 3.0 mg/kg pifithrin-alpha or 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha after pretreatment with the selective PI3K inhibitor wortmannin (0.6 mg/kg) or the mitochondrial permeability transition pore opener atractyloside (5 mg/kg). RESULTS: Isoflurane (1.0 but not 0.5 MAC), pifithrin-alpha (3.0 but not 1.5 mg/kg), and the combination of 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha significantly (P < 0.05) reduced infarct size (21% +/- 4%, 43% +/- 7%, 22% +/- 4%, 45% +/- 4%, and 28% +/- 3% [mean +/- sd], respectively, of left ventricular area at risk; triphenyltetrazolium chloride staining) when compared with control (45% +/- 2%). Atractyloside, but not wortmannin, abolished 3.0 mg/kg pifithrin-alpha-induced cardioprotection, whereas atractyloside and wortmannin blocked reductions in infarct size produced by 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha. CONCLUSION: The results indicate that inhibition of the apoptotic protein p53 lowers the threshold of isoflurane-induced cardioprotection during early reperfusion in vivo.

Rosiglitazone antagonizes vascular endothelial growth factor signaling and nuclear factor of activated T cells activation in cardiac valve endothelium.

Nuclear factor of activated T cells, Cytoplasmic 1 (NFATc1) is required for heart valve formation. Vascular endothelial growth factor (VEGF) signaling, mediated by NFATc1 activation, positively regulates growth of valvular endothelial cells. However, regulators of VEGF/NFATc1 signaling in valve endothelium are poorly understood. Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits NFATc1 activity in T cells and cardiomyocytes, but it is not known if PPARgamma controls NFATc1 function in endothelial cells. The authors hypothesize PPARgamma antagonizes VEGF signaling in valve endothelium by inhibiting NFATc1. Endothelial cells isolated from human valve leaflet tissue were shown by immunocytochemistry to express the endothelial-specific markers von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule (PECAM)-1. VEGF-induced proliferation and migration of human pulmonary valve endothelial cells (HPVECs) were inhibited by rosiglitazone (ROSI), a specific ligand of PPARgamma activation, suggesting that PPARgamma disrupts VEGF signaling in the valve endothelium. ROSI also antagonized VEGF-mediated NFATc1 nuclear translocation in HPVECs, suggesting that PPARgamma inhibits VEGF signaling of NFATc1 activation in the valve. The effect of ROSI on nonvalve human umbilical vein endothelial cells (HUVECs) was tested in parallel and a similar inhibition of NFATc1 activation was observed. These data provide the first demonstration that ROSI negatively regulates VEGF signaling in the valve endothelium by a mechanism involving NFATc1 activation and nuclear translocation.

Extracellular signal-regulated kinases trigger isoflurane preconditioning concomitant with upregulation of hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in rats.

INTRODUCTION: Extracellular signal-related kinases 1 and 2 (Erk1/2) are mitogen-activated protein kinases that have been implicated in anesthetic preconditioning; but whether Erk1/2 triggers or mediates this beneficial effect and the mechanisms by which Erk1/2 produces cardioprotection are unknown. We tested the hypothesis that isoflurane preconditioning is triggered by Erk1/2 concomitant with upregulation of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression in rats instrumented for hemodynamic measurement and subjected to a 30-min coronary artery occlusion and 2-h reperfusion. METHODS: Rats randomly received IV 0.9% saline (control) or isoflurane (1.0 minimum alveolar concentration administered for 30 min and discontinued 15 min [memory period] before coronary occlusion) in the absence or presence of the selective Erk1/2 inhibitor PD 098059 (1 mg/kg in dimethylsulfoxide administered IV either 3 min before exposure to isoflurane [trigger] or 3 min after discontinuation of the drug [mediator]). Additional rabbits were pretreated with dimethylsulfoxide alone. Left ventricular tissue samples were obtained at selected intervals from additional groups of rats for Western immunoblot analysis of phospho-Erk1/2, HIF-1alpha, and VEGF protein expression. RESULTS: Isoflurane significantly (P < 0.05) reduced infarct size (41% +/- 8% of the left ventricular area at risk; triphenyltetrazolium chloride staining) as compared with control (59% +/- 4%). PD 098059 administered before, but not after, isoflurane abolished this cardioprotection (61% +/- 5% and 42% +/- 9%, respectively). Isoflurane-induced increases in phospho-Erk1/2, HIF-1alpha, and VEGF expression were also inhibited by PD 098059 pretreatment. CONCLUSIONS: The results indicate that Erk1/2 triggers isoflurane preconditioning concomitant with HIF-1alpha and VEGF upregulation in vivo.