Decrease in Plasma miR-27a and miR-221 After Concussion in Australian Football Players.

INTRODUCTION: Sports-related concussion (SRC) is a common form of brain injury that lacks reliable methods to guide clinical decisions. MicroRNAs (miRNAs) can influence biological processes involved in SRC, and measurement of miRNAs in biological fluids may provide objective diagnostic and return to play/recovery biomarkers. Therefore, this prospective study investigated the temporal profile of circulating miRNA levels in concussed male and female athletes. METHODS: Pre-season baseline blood samples were collected from amateur Australian rules football players (82 males, 45 females). Of these, 20 males and 8 females sustained an SRC during the subsequent season and underwent blood sampling at 2-, 6- and 13-days post-injury. A miRNA discovery Open Array was conducted on plasma to assess the expression of 754 known/validated miRNAs. miRNA target identified were further investigated with quantitative real-time PCR (qRT-PCR) in a validation study. Data pertaining to SRC symptoms, demographics, sporting history, education history and concussion history were also collected. RESULTS: Discovery analysis identified 18 candidate miRNA. The consequent validation study found that plasma miR-221-3p levels were decreased at 6d and 13d, and that miR-27a-3p levels were decreased at 6d, when compared to baseline. Moreover, miR-27a and miR-221-3p levels were inversely correlated with SRC symptom severity. CONCLUSION: Circulating levels of miR-27a-3p and miR-221-3p were decreased in the sub-acute stages after SRC, and were inversely correlated with SRC symptom severity. Although further studies are required, these analyses have identified miRNA biomarker candidates of SRC severity and recovery that may one day assist in its clinical management.

Oral fucoidan improves muscle size and strength in mice.

Fucoidan is a sulfated polysaccharide found in a range of brown algae species. Growing evidence supports the long-term supplementation of fucoidan as an ergogenic aid to improve skeletal muscle performance. The aim of this study was to investigate the effect of fucoidan on the skeletal muscle of mice. Male BL/6 mice (N = 8-10) were administered a novel fucoidan blend (FUC, 400 mg/kg/day) or vehicle (CON) for 4 weeks. Treatment and control experimental groups were further separated into exercise (CON+EX, FUC+EX) or no-exercise (CON, FUC) groups, where exercised groups performed 30 min of treadmill training three times per week. At the completion of the 4-week treatment period, there was a significant increase in cross-sectional area (CSA) of muscle fibers in fucoidan-treated extensor digitorum longus (EDL) and soleus fibers, which was accompanied by a significant increase in tibialis anterior (TA) muscle force production in fucoidan-treated groups. There were no significant changes in grip strength or treadmill time to fatigue, nor was there an effect of fucoidan or exercise on mass of TA, EDL, or soleus muscles. In gastrocnemius muscles, there was no change in mRNA expression of mitochondrial biogenesis markers PGC-1α and Nrf-2 in any experimental groups; however, there was a significant effect of fucoidan supplementation on myosin heavy chain (MHC)-2x, but not MHC-2a, mRNA expression. Overall, fucoidan increased muscle size and strength after 4 weeks of supplementation in both exercised and no-exercised mice suggesting an important influence of fucoidan on skeletal muscle physiology.

A Comparison of the Gluco-Regulatory Responses to High-Intensity Interval Exercise and Resistance Exercise.

High-intensity interval exercise and resistance exercise both effectively lower blood glucose; however, it is not clear whether different regulatory mechanisms exist. This randomised cross-over study compared the acute gluco-regulatory and the physiological responses of high-intensity interval exercise and resistance exercise. Sixteen (eight males and eight females) recreationally active individuals, aged (mean ± SD) 22 ± 7 years, participated with a seven-day period between interventions. The high-intensity interval exercise trial consisted of twelve, 30 s cycling intervals at 80% of peak power capacity and 90 s active recovery. The resistance exercise trial consisted of four sets of 10 repetitions for three lower-limb exercises at 80% 1-RM, matched for duration of high-intensity interval exercise. Exercise was performed after an overnight fast, with blood samples collected every 30 min, for two hours after exercise. There was a significant interaction between time and intervention for glucose (p = 0.02), which was, on average (mean ± SD), 0.7 ± 0.7 mmol∙L-1 higher following high-intensity interval exercise, as compared to resistance exercise. Cortisol concentration over time was affected by intervention (p = 0.03), with cortisol 70 ± 103 ng∙mL-1 higher (p = 0.015), on average, following high-intensity interval exercise. Resistance exercise did not induce the acute rise in glucose that was induced by high-intensity interval exercise and appears to be an appropriate alternative to positively regulate blood glucose.

Gambogic amide, a selective TrkA agonist, does not improve outcomes from traumatic brain injury in mice.

OBJECTIVES: There is evidence that treatment with nerve growth factor (NGF) may reduce neuroinflammation and apoptosis after a traumatic brain injury (TBI). NGF is thought to exert its effects via binding to either TrkA or p75 neurotrophin receptors. This study aimed to investigate the effects of a selective TrkA agonist, gambogic amide (GA), on TBI pathology and outcomes in mice following lateral fluid percussion injury. METHODS: Male C57BL/6 mice were given either a TBI or sham injury, and then received subcutaneous injections of either 2 mg/kg of GA or vehicle at 1, 24, and 48 h post-injury. Following behavioural studies, mice were euthanized at 72 h post-injury for analysis of neuroinflammatory, apoptotic, and neurite outgrowth markers. RESULTS: Behavioural testing revealed that GA did not mitigate motor deficits after TBI. TBI caused an increase in cortical and hippocampal expression of several markers of neuroinflammation and apoptosis compared to sham groups. GA treatment did not attenuate these increases in expression, possibly contributed to by our finding of TrkA receptor down-regulation post-TBI. CONCLUSIONS: These findings suggest that GA treatment may not be suitable for attenuating TBI pathology and improving outcomes.

Ion transporters in brain endothelial cells that contribute to formation of brain interstitial fluid.

Ions and water transported across the endothelium lining the blood­brain barrier contribute to the fluid secreted into the brain and are important in maintaining appropriate volume and ionic composition of brain interstitial fluid. Changes in this secretion process may occur after stroke. The present study identifies at transcript and protein level ion transporters involved in the movement of key ions and examines how levels of certain of these alter following oxidative stress. Immunohistochemistry provides evidence for Cl−/HCO3− exchanger, AE2, and Na+, HCO3− cotransporters, NBCe1 and NBCn1, on brain microvessels. mRNA analysis by RT-PCR reveals expression of these transporters in cultured rat brain microvascular endothelial cells (both primary and immortalized GPNT cells) and also Na+/H+ exchangers, NHE1 (primary and immortalized) and NHE2 (primary cells only). Knock-down using siRNA in immortalized GPNT cells identifies AE2 as responsible for much of the Cl−/HCO3− exchange following extracellular chloride removal and NHE1 as the transporter that accounts for most of the Na+/H+ exchange following intracellular acidification. Transcript levels of both AE2 and NHE1 are increased following hypoxia/reoxygenation. Further work is now required to determine the localization of the bicarbonate transporters to luminal or abluminal membranes of the endothelial cells as well as to identify and localize additional transport mechanisms that must exist for K+ and Cl−.

Circulating non-coding RNAs as biomarkers of beta cell death in diabetes.

Death of pancreatic islet beta cells is a common feature of type 1 and 2 diabetes and often follows islet cell transplantation. Measurement of blood glucose is currently the only blunt instrument available to diagnose diabetes mellitus, and we lack tools to quantify islet cell loss or protection thereof. A class of RNA molecules (called microRNAs/miRNAs/miRs) that regulate endogenous gene expression via mRNA cleavage or translational arrest have been identified to be critical for birth, maintenance and regeneration of pancreatic beta cells. Recent demonstration that microRNAs can potentially be utilised as biomarkers due to their serum stability, has triggered increasing interest in understanding their role as regulators or biomarkers of disease. This review aims to delve into the potential of miRNA biomarkers, and whether miRNA profiles are indicators or effector of disease pathology. Furthermore, an outline for identifying and confirming islet-specific miRNA biomarkers is discussed.

Annexin peptide Ac2-26 suppresses TNFα-induced inflammatory responses via inhibition of Rac1-dependent NADPH oxidase in human endothelial cells.

The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNFα) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2',7'-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NFκB) was measured by luciferase activity assay. TNFα stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5-1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNFα-induced NFκB promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNFα whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNFα-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNFα-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNFα-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NFκB pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.

NADPH oxidase and angiogenesis following endothelin-1 induced stroke in rats: role for nox2 in brain repair.

NADPH oxidases contribute to brain injury, yet they may also have a role in brain repair, particularly in vascular signaling and angiogenesis. This study determined the temporal and spatial profile of NADPH oxidase subunit expression/activity concurrently with angiogenesis in the brain following transient ischemic stroke induced by prolonged constriction of the middle cerebral artery by perivascular injection of endothelin-1 in conscious Hooded Wistar rats (n = 47). VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days. Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days. Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed. These results indicate for the first time that ROS signaling through a cerebrovascular Nox2 NADPH oxidase may be important in initiating brain angiogenesis.

A protocol for measurement of noncoding RNA in human serum.

MicroRNAs (miRNAs) are small noncoding RNAs that act as regulators of gene expression by targeting mature messenger RNAs. Following the initial report of the presence of miRNAs in serum and plasma a number of studies have successfully demonstrated the use of these miRNAs as biomarkers of disease. Currently, there are many methods of isolating total RNA from liquid samples. Here, we describe a simple, cost effective method for extraction of RNA from human serum as well as subsequent real time PCR analysis of miRNA levels.

The in vitro preconditioning of myoblasts to enhance subsequent survival in an in vivo tissue engineering chamber model.

The effects of in vitro preconditioning protocols on the ultimate survival of myoblasts implanted in an in vivo tissue engineering chamber were examined. In vitro testing: L6 myoblasts were preconditioned by heat (42 °C; 1.5 h); hypoxia (<8% O(2); 1.5 h); or nitric oxide donors: S-nitroso-N-acetylpenicillamine (SNAP, 200 µM, 1.5 h) or 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NONOate, 500 µM, 7 h). Following a rest phase preconditioned cells were exposed to 24 h hypoxia, and demonstrated minimal overall cell loss, whilst controls (not preconditioned, but exposed to 24 h hypoxia) demonstrated a 44% cell loss. Phosphoimmunoblot analysis of pro-survival signaling pathways revealed significant activation of serine threonine kinase Akt with DETA-NONOate (p < 0.01) and heat preconditioning (p < 0.05). DETA-NONOate also activated ERK 1/2 signaling (p < 0.05). In vivo implantation: 100,000 preconditioned (heat, hypoxia, or DETA-NONOate) myoblasts were implanted in SCID mouse tissue engineering chambers. 100,000 (not preconditioned) myoblasts were implanted in control chambers. At 3 weeks, morphometric assessment of surviving myoblasts indicated myoblast percent volume (p = 0.012) and myoblasts/mm(2) (p = 0.0005) overall significantly increased in preconditioned myoblast chambers compared to control, with DETA-NONOate-preconditioned myoblasts demonstrating the greatest increase in survival (p = 0.007 and p = 0.001 respectively). DETA-NONOate therefore has potential therapeutic benefits to significantly improve survival of transplanted cells.

Prostacyclin receptor suppresses cardiac fibrosis: role of CREB phosphorylation.

Cardiac fibrosis is a consequence of many cardiovascular diseases and contributes to impaired ventricular function. Activation of the prostacyclin receptor (IP) protects against cardiac fibrosis, but the molecular mechanisms are not totally understood. Using mouse cardiac fibroblasts, we found that IP activation with cicaprost suppressed expression of collagen I and other target genes of transforming growth factor-beta. This effect of cicaprost was unlikely to be mediated by inhibition of the Smad2/3 or mitogen-activated protein kinase (MAPK) activities, but was associated with cAMP elevation and phosphorylation of the transcription factor cAMP response element binding protein (CREB). Expression of a non-phosphorylated CREB mutant suppressed the inhibitory effect of cicaprost. It appears that phosphorylated CREB binds to and sequestrates the transcription coactivator CBP/p300 from binding to Smad. Inhibition of the intrinsic histone acetyl-transferase activity of CBP/p300 with garcinol significantly suppressed collagen I expression in fibroblasts. Using apolipoprotein E and IP double knockout mouse, we demonstrated that endogenous prostacyclin/IP signaling had an inhibitory effect on angiotensin II-induced cardiac fibrosis under hypercholesterolemic conditions. Taken together, our results suggest that the prostacyclin/IP pathway suppresses cardiac fibrosis, at least partly, by inducing CREB phosphorylation.

Translation-linked mRNA destabilization accompanying serum-induced Nox4 expression in human endothelial cells.

NADPH oxidase is involved in cell signaling, regulating proliferation of vascular cells, especially in endothelium. The Nox4 catalytic subunit has a major role in endothelial cells, but growth arrest of cultured endothelial cells following serum deprivation paradoxically increases mRNA for Nox4. We investigated the relationships between Nox4 mRNA stability and protein expression in human microvascular endothelial cells. Serum starvation increased the steady-state level of Nox4 mRNA but paradoxically diminished Nox4 protein expression. mRNA transcription in the absence of serum is maintained by the p38MAP kinase pathway, for inhibition of p38MAP kinase reduced both Nox4 mRNA and Nox4 promoter activity. In serum-starved cells, reintroduction of serum increased Nox4 protein levels within 30 min and up to 24 h. In contrast, the mRNA decreased equally rapidly after serum stimulation. Inhibition of Nox4 translation by cycloheximide blocked serum-induced mRNA degradation and Nox4 protein synthesis, and actinomycin-D also delayed Nox4 mRNA decay. Therefore, Nox4 mRNA level falls after serum stimulation because of a translation-initiated mRNA destabilization program. Clearly Nox4 mRNA is regulated at both transcriptional and post-transcriptional levels, and the steady state level of Nox4 mRNA does not accurately reflect Nox4 protein abundance and functions, with implications for regulation of cell proliferation and survival.

Transport activities involved in intracellular pH recovery following acid and alkali challenges in rat brain microvascular endothelial cells.

Transport activities involved in intracellular pH (pH(i)) recovery after acid or alkali challenge were investigated in cultured rat brain microvascular endothelial cells by monitoring pH(i) using a pH-sensitive dye. Following relatively small acid loads with pH(i) approximately 6.5, HCO(-)(3) influx accounted for most of the acid extrusion from the cell with both Cl(-)-independent and Cl(-)-dependent, Na(+)-dependent transporters involved. The Cl(-)-independent component has the same properties as the NBC-like transporter previously shown to account for most of the acid extrusion near the resting pH(i). Following large acid loads with pH(i) < 6.5, most of the acid extrusion was mediated by Na(+)/H(+) exchange, the rate of which was steeply dependent on pH(i). Concanamycin A, an inhibitor of V-type ATPase, had no effect on the rates of acid extrusion. Following an alkali challenge, the major component of the acid loading leading to recovery of pH(i) occurred by Cl(-)/HCO(-)(3) exchange. This exchange had the same properties as the AE-like transporter previously identified as a major acid loader near resting pH(i). These acid-loading and acid-extruding transport mechanisms together with the Na(+), K(+), ATPase may be sufficient to account not only for pH(i) regulation in brain endothelial cells but also for the net secretion of HCO(-)(3) across the blood-brain barrier.

Induction of heme oxygenase-1 in vivo suppresses NADPH oxidase derived oxidative stress.

Our previous studies suggest that heme oxygenase (HO)-1 induction and/or subsequent bilirubin generation in endothelial cells may suppress superoxide generation of from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. In this study, we examined the consequence of HO-1 induction in vivo on NADPH oxidase activity. Three doses of hemin (25 mg x kg(-1), IP, every 48 hours), with or without cotreatment with the HO inhibitor tin protoporphyrin-IX (15 mg x kg(-1), IP), were given to apolipoprotein E-deficient mice, which display vascular oxidative stress. Hemin treatment increased HO-1 expression and activity in aorta (undetectable at baseline) and kidney (by 3-fold) and significantly reduced both NADPH oxidase activity (by approximately 25% to 50%) and superoxide generation in situ. The increase in HO-1 activity and inhibition of NADPH oxidase activity by hemin were reversed by tin protoporphyrin-IX and were not associated with changes in Nox2 or Nox4 protein levels. Hemin also reduced plasma F(2)-isoprostane levels by 23%. The inhibition of NADPH oxidase activity by hemin in the aorta was mimicked by bilirubin in vitro (0.01 to 1 micromol/L). Bilirubin also concentration-dependently reduced NADPH oxidase-dependent superoxide production stimulated by angiotensin II in rat vascular smooth muscle cells and by phorbol 12-myristate 13-acetate in human neutrophil-like HL-60 cells. HO-1 overexpression by plasmid-mediated gene transfer in rat vascular smooth muscle cells decreased NADPH-stimulated superoxide production. Thus, systemic expression of HO-1 suppresses NADPH oxidase activity by mechanisms at least partly mediated by the bile pigment bilirubin, thereby reducing oxidative stress.

Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells.

Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO(3)(-), which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH(i)). To analyse the transporters that may be involved in the maintenance of pH(i) and hence secretion of HCO(3)(-), we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO(2)/HCO(3)(-). pH(i) in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO(3)(-) than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO(3)(-) were similar. In the nominal absence of HCO(3)(-) the rate of acid loading was increased equally by removal of external Na(+) or by inhibition of Na(+)/H(+) exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO(3)(-) the increase in the rate of acid loading when Na(+) was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na(+) and HCO(3)(-). Removal of Cl(-) in the presence of HCO(3)(-) produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na(+) following removal of Cl(-) increased the rate of acid gain. In the presence of HCO(3)(-) and initial presence of Na(+) and Cl(-), DIDS inhibited the changes in pH(i) produced by removal of either Na(+) or Cl(-). These are the expected results if these cells possess an AE-like Cl(-)/HCO(3)(-) exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO(3)(-)), a NBC-like Cl(-)-independent Na(+)-HCO(3)(-) cotransporter, and a NHE-like Na(+)/H(+) exchanger. The in vitro rates of HCO(3)(-) loading via the Na(+)-HCO(3)(-) cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO(3)(-) into the brain.

Protein kinase C and downstream signaling pathways in a three-dimensional model of phorbol ester-induced angiogenesis.

Angiogenesis, a critical process in both health and disease, is mediated by a number of signaling pathways. Although proangiogenic stimuli, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and the phorbol ester phorbol-12 myristate-13 acetate (PMA) are known to promote blood vessel formation, their downstream targets are ill defined. We sought to investigate the signaling pathways required for vessel assembly by utilizing a three-dimensional collagen matrix in which human umbilical vein endothelial cells (HUVECs) form tubular structures. Our data show that PMA is sufficient for the induction of angiogenesis, and that protein kinase C (PKC) is necessary for this process. Evaluation of PKC isoforms alpha and sigma revealed that these proteins are uniquely regulated. Characterization of an additional PMA target, protein kinase D (PKD) demonstrated that this enzyme becomes phosphorylated in HUVECs, and may therefore be involved in proangiogenic signaling. Further examination of downstream effectors of PKC showed that extracellular signal-regulated kinase (ERK) is critical for angiogenesis, and is accordingly phosphorylated in response to PMA. Surprisingly however, phosphorylation of ERK is independent of PKC activity. In addition, we show that the PKC target sphingosine kinase (SPK) is required for vessel formation. These findings illustrate the complexities of blood vessel formation, and suggest that activators utilize multiple independent pathways to invoke a complete angiogenic response.

Hypoxia-inducible factor 1alpha modulates adhesion, migration, and FAK phosphorylation in vascular smooth muscle cells.

Hypoxia promotes angiogenesis by modulating the transcriptional regulator hypoxia-inducible factor 1alpha (HIF-1alpha). HIF-1alpha is a master regulator of the hypoxic response, and its proangiogenic activities include, but are not limited to, regulation of vascular endothelial growth factor (VEGF). The remodeling of the vasculature during angiogenesis requires an initial destabilization step, which facilitates endothelial sprouting, followed by vessel growth, and restabilization through investment of smooth muscle cells. The complex dynamics of hypoxia-induced angiogenesis prompted us to investigate what aspects of this multi-step process are regulated by HIF-1alpha. To do so, we analyzed the molecular properties of aortic and coronary artery smooth muscle cells in response to forced expression of HIF-1alpha, and by treatment with cobalt chloride, which mimics hypoxia. Our results demonstrate that HIF-1alpha causes a marked reduction in the ability of smooth muscle cells to migrate and adhere to extracellular matrices. Analysis of focal adhesion proteins showed no significant difference in expression or localization of vinculin or focal adhesion kinase (FAK). However, investigation of FAK phosphorylation, a critical mediator of adhesion and migration, revealed tyrosine phosphorylation of FAK is diminished in the presence of HIF-1alpha and cobalt chloride. These results indicate that during hypoxia-induced vessel remodeling, HIF-1alpha functions to dampen adhesion and migration of smooth muscle cells by modulating FAK activity. We suggest that HIF-1alpha expression in smooth muscle cells may augment vessel sprouting by loosening smooth muscle cell attachments to the basement membrane and endothelial cells.