FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury.

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron, with alterations in associated transcription factors detected both locally and remote to the lesion. Stress-induced nuclear translocation of the transcription factor forkhead class box O3a (FOXO3a) was initially linked to activation of apoptotic genes in many neuronal subtypes. However, a more complex role of FOXO3a has been suggested in the injury response of sensory neurons, with the injured neuron expressing less FOXO3a. To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression, the temporal impact of chronic unilateral L4-6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral, contralateral or remote to injury relative to naïve controls was examined. In naïve neurons, high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide, a marker of the nociceptive subpopulation. One hour post-injury, an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1, 2 and 4 days, with levels increasing toward pre-injury levels by 1 week post-injury. A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury. Nuclear levels of FOXO3a peaked at 1 day, decreased by 4 days, then increased by 1 week post-injury, a response mirrored in C4 dorsal root ganglion neurons remote to injury. This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts, a response we recently reported for another stress transcription factor, Luman/CREB3. The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective. Finally, the broader systemic changes support the existence of stress/injury-induced humeral factor(s) influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons. Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board (protocol #19920164).

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis.

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.

Distal coronary embolization following acute myocardial infarction increases early infarct size and late left ventricular wall thinning in a porcine model.

BACKGROUND: Distal coronary embolization (DCE) of thrombotic material occurs frequently during percutaneous interventions for acute myocardial infarction and can alter coronary flow grades. The significance of DCE on infarct size and myocardial function remains unsettled. The aims of this study were to evaluate the effects of DCE sufficient to cause no-reflow on infarct size, cardiac function and ventricular remodeling in a porcine acute myocardial infarction model. METHODS AND RESULTS: Female Yorkshire pigs underwent 60 min balloon occlusion of the left anterior descending coronary artery followed by reperfusion and injection of either microthrombi (prepared from autologous porcine blood) sufficient to cause no-reflow (DCE), or saline (control). Animals were sacrificed at 3 h (n = 5), 3 days (n = 20) or 6 weeks (n = 20) post-AMI. Cardiovascular magnetic resonance (CMR), serum troponin-I, and cardiac gelatinase (MMP) and survival kinase (Akt) activities were assessed. At 3d, DCE increased infarct size (CMR: 18.8% vs. 14.5%, p = 0.04; serum troponin-I: 13.3 vs. 6.9 ng/uL, p < 0.05) and MMP-2 activity levels (0.81 vs. 0.49, p = 0.002), with reduced activation of Akt (0.06 versus 0.26, p = 0.02). At 6 weeks, there were no differences in infarct size, ventricular volume or ejection fraction between the two groups, although infarct transmurality (70% vs. 57%, p< 0.04) and ventricular thinning (percent change in mid anteroseptal wall thickness:-25.6% vs. 0.7%, p = 0.03) were significantly increased in the DCE group. CONCLUSIONS: DCE increased early infarct size, but without affecting later infarct size, cardiac function or ventricular volumes. The significance of the later remodelling changes (ventricular thinning and transmurality) following DCE, possibly due to changes in MMP-2 activity and Akt activation, merits further study.

Cardioprotective Signature of Short-Term Caloric Restriction.

OBJECTIVE: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR). BACKGROUND: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia. METHODS: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI. RESULTS: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

Perlecan heparan sulfate proteoglycan is a critical determinant of angiogenesis in response to mouse hind-limb ischemia.

BACKGROUND: Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown. METHODS: Heparan sulfate (HS)-deficient perlecan (Hspg2(Δ3/Δ3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF)2 protein expression at days 2 and 28. RESULTS: Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Δ3/Δ3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Δ3/Δ3) mice at day 28 (0.67 ± 0.12 vs 0.26 ± 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Δ3/Δ3) mice on day 28 (122 ± 30 cells vs 84 ± 34 cells per high-power field [HPF]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) were significantly decreased in the ischemic limbs of Hspg2(Δ3/Δ3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences. CONCLUSIONS: These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.

p27 protein protects metabolically stressed cardiomyocytes from apoptosis by promoting autophagy.

p27(Kip1) (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo.

Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection.

The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX(-/-)) model and found that survival after infection was significantly greater in GX(-/-) mice than in GX(+/+) mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX(-/-) mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX(-/-) mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza.