Ascorbate insufficiency disrupts glutamatergic signaling and alters electroencephalogram phenotypes in a mouse model of Alzheimer's disease.

Clinical studies have reported that increased epileptiform and subclinical epileptiform activity can be detected in many patients with an Alzheimer's disease (AD) diagnosis using electroencephalogram (EEG) and this may correlate with poorer cognition. Ascorbate may have a specific role as a neuromodulator in AD as it is released concomitantly with glutamate reuptake following excitatory neurotransmission. Insufficiency may therefore result in an exacerbated excitatory/inhibitory imbalance in neuronal signaling. Using a mouse model of AD that requires dietary ascorbate (Gulo-/-APPswe/PSEN1dE9), EEG was recorded at baseline and during 4 weeks of ascorbate depletion in young (5-month-old) and aged (20-month-old) animals. Data were scored for changes in quantity of spike trains, individual spikes, sleep-wake rhythms, sleep fragmentation, and brainwave power bands during light periods each week. We found an early increase in neuronal spike discharges with age and following ascorbate depletion in AD model mice and not controls, which did not correlate with brain amyloid load. Our data also show more sleep fragmentation with age and with ascorbate depletion. Additionally, changes in brain wave activity were observed within different vigilance states in both young and aged mice, where Gulo-/-APPswe/PSEN1dE9 mice had shifts towards higher frequency bands (alpha, beta, and gamma) and ascorbate depletion resulted in shifts towards lower frequency bands (delta and theta). Microarray data supported ascorbate insufficiency altering glutamatergic transmission through the decreased expression of glutamate related genes, however no changes in protein expression of glutamate reuptake transporters were observed. These data suggest that maintaining optimal brain ascorbate levels may support normal brain electrical activity and sleep patterns, particularly in AD patient populations where disruptions are observed.

Customisable Silicone Vessels and Tissue Phantoms for In Vitro Photoplethysmography Investigations into Cardiovascular Disease.

Age-related vessel deterioration leads to changes in the structure and function of the heart and blood vessels, notably stiffening of vessel walls, increasing the risk of developing cardiovascular disease (CVD), which accounts for 17.9 million global deaths annually. This study describes the fabrication of custom-made silicon vessels with varying mechanical properties (arterial stiffness). The primary objective of this study was to explore how changes in silicone formulations influenced vessel properties and their correlation with features extracted from signals obtained from photoplethysmography (PPG) reflectance sensors in an in vitro setting. Through alterations in the silicone formulations, it was found that it is possible to create elastomers exhibiting an elasticity range of 0.2 MPa to 1.22 MPa. It was observed that altering vessel elasticity significantly impacted PPG signal morphology, particularly reducing amplitude with increasing vessel stiffness (p < 0.001). A p-value of 5.176 × 10-15 and 1.831 × 10-14 was reported in the red and infrared signals, respectively. It has been concluded in this study that a femoral artery can be recreated using the silicone material, with the addition of a softener to achieve the required mechanical properties. This research lays the foundation for future studies to replicate healthy and unhealthy vascular systems. Additional pathologies can be introduced by carefully adjusting the elastomer materials or incorporating geometrical features consistent with various CVDs.

Photoplethysmography for the Assessment of Arterial Stiffness.

This review outlines the latest methods and innovations for assessing arterial stiffness, along with their respective advantages and disadvantages. Furthermore, we present compelling evidence indicating a recent growth in research focused on assessing arterial stiffness using photoplethysmography (PPG) and propose PPG as a potential tool for assessing vascular ageing in the future. Blood vessels deteriorate with age, losing elasticity and forming deposits. This raises the likelihood of developing cardiovascular disease (CVD), widely reported as the global leading cause of death. The ageing process induces structural modifications in the vascular system, such as increased arterial stiffness, which can cause various volumetric, mechanical, and haemodynamic alterations. Numerous techniques have been investigated to assess arterial stiffness, some of which are currently used in commercial medical devices and some, such as PPG, of which still remain in the research space.

Effects of Contact Pressure in Reflectance Photoplethysmography in an In Vitro Tissue-Vessel Phantom.

With the continued development and rapid growth of wearable technologies, PPG has become increasingly common in everyday consumer devices such as smartphones and watches. There is, however, minimal knowledge on the effect of the contact pressure exerted by the sensor device on the PPG signal and how it might affect its morphology and the parameters being calculated. This study explores a controlled in vitro study to investigate the effect of continually applied contact pressure on PPG signals (signal-to-noise ratio (SNR) and 17 morphological PPG features) from an artificial tissue-vessel phantom across a range of simulated blood pressure values. This experiment confirmed that for reflectance PPG signal measurements for a given anatomical model, there exists an optimum sensor contact pressure (between 35.1 mmHg and 48.1 mmHg). Statistical analysis shows that temporal morphological features are less affected by contact pressure, lending credit to the hypothesis that for some physiological parameters, such as heart rate and respiration rate, the contact pressure of the sensor is of little significance, whereas the amplitude and geometric features can show significant change, and care must be taken when using morphological analysis for parameters such as SpO2 and assessing autonomic responses.

Loss of 2 Akt (Protein Kinase B) Isoforms in Hematopoietic Cells Diminished Monocyte and Macrophage Survival and Reduces Atherosclerosis in Ldl Receptor-Null Mice.

Objective- Macrophages express 3 Akt (protein kinase B) isoforms, Akt1, Akt2, and Akt3, which display isoform-specific functions but may be redundant in terms of Akt survival signaling. We hypothesize that loss of 2 Akt isoforms in macrophages will suppress their ability to survive and modulate the development of atherosclerosis. Approach and Results- To test this hypothesis, we reconstituted male Ldlr-/- mice with double Akt2/Akt3 knockout hematopoietic cells expressing only the Akt1 isoform (Akt1only). There were no differences in body weight and plasma lipid levels between the groups after 8 weeks of the Western diet; however, Akt1only→ Ldlr-/- mice developed smaller (57.6% reduction) atherosclerotic lesions with more apoptotic macrophages than control mice transplanted with WT (wild type) cells. Next, male and female Ldlr-/- mice were reconstituted with double Akt1/Akt2 knockout hematopoietic cells expressing the Akt3 isoform (Akt3only). Female and male Akt3only→ Ldlr-/- recipients had significantly smaller (61% and 41%, respectively) lesions than the control WT→ Ldlr-/- mice. Loss of 2 Akt isoforms in hematopoietic cells resulted in markedly diminished levels of white blood cells, B cells, and monocytes and compromised viability of monocytes and peritoneal macrophages compared with WT cells. In response to lipopolysaccharides, macrophages with a single Akt isoform expressed low levels of inflammatory cytokines; however, Akt1only macrophages were distinct in expressing high levels of antiapoptotic Il10 compared with WT and Akt3only cells. Conclusions- Loss of 2 Akt isoforms in hematopoietic cells, preserving only a single Akt1 or Akt3 isoform, markedly compromises monocyte and macrophage viability and diminishes early atherosclerosis in Ldlr-/- mice.

Novel Polydimethylsiloxane (PDMS) Pulsatile Vascular Tissue Phantoms for the In-Vitro Investigation of Light Tissue Interaction in Photoplethysmography.

Currently there exists little knowledge or work in phantoms for the in-vitro evaluation of photoplethysmography (PPG), and its' relationship with vascular mechanics. Such phantoms are needed to provide robust, basic scientific knowledge, which will underpin the current efforts in developing new PPG technologies for measuring or estimating blood pressure, blood flow and arterial stiffness, to name but a few. This work describes the design, fabrication and evaluation of finger tissue-simulating pulsatile phantoms with integrated custom vessels. A novel technique has been developed to produce custom polydimethylsiloxane (PDMS) vessels by a continuous dip-coating process. This process can accommodate the production of different sized vessel diameters (1400-2500 µm) and wall thicknesses (56-80 µm). These vessels were embedded into a mould with a solution of PDMS and India ink surrounding them. A pulsatile pump experimental rig was set up to test the phantoms, where flow rate (1-12 L·min-1), heart rate (40-120 bpm), and total resistance (0-100% resistance clamps) could be controlled on demand. The resulting flow profiles approximates human blood flow, and the detected contact PPG signal (red and infrared) from the phantom closely resembles the morphology of in-vivo PPG waveforms with signal-to-noise ratios of 38.16 and 40.59 dB, for the red and infrared wavelengths, respectively. The progress made by this phantom development will help in obtaining new knowledge in the behaviour of PPG's under differing flow conditions, optical tissue properties and differing vessel stiffness.

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin.

BACKGROUND: Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity. METHODS: Human umbilical vein endothelial cells (HUVEC) were grown to confluence and treated with CFH with or without ascorbate. Monolayer permeability was measured by Electric Cell-substrate Impedance Sensing (ECIS) or transfer of 14C-inulin. Viability was measured by trypan blue exclusion. Intracellular ascorbate was measured by HPLC. RESULTS: CFH increased permeability in a dose- and time-dependent manner with 1 mg/ml of CFH increasing inulin transfer by 50% without affecting cell viability. CFH (1 mg/ml) also caused a dramatic reduction in intracellular ascorbate in the same time frame (1.4 mM without CFH, 0.23 mM 18 h after 1 mg/ml CFH, p < 0.05). Pre-treatment of HUVECs with ascorbate attenuated CFH induced permeability. CONCLUSIONS: CFH increases endothelial permeability in part through depletion of intracellular ascorbate. Supplementation of ascorbate can attenuate increases in permeability mediated by CFH suggesting a possible therapeutic approach in sepsis.

Effect of Impact Mechanism on Head Accelerations in Men's Lacrosse Athletes.

Quantifying head impacts is a vital component to understanding and preventing head trauma in sport. Our objective was to establish the frequency and magnitude of head impact mechanisms in men's lacrosse athletes. Eleven male lacrosse athletes wore xPatch sensors during activity. Video footage of practices and games was analyzed to verify impacts and code them with impact mechanisms. The authors calculated incidence rates (IRs) per 1000 exposures with corresponding 95% confidence intervals (CIs) and used multivariate analysis of variances to compare the linear (g) and rotational (rad/s2) accelerations between mechanisms. A total of 167 head impacts were successfully verified and coded with a mechanism using video footage during 542 total exposures. The highest IR was head to body (IR = 118.08; 95% CI, 89.15-147.01), and the lowest was head to ball (IR = 3.69; 95% CI, 0-8.80) (incidence rate ratio = 32.00; 95% CI, 67.83-130.73). Analysis indicated that impact mechanism failed to significantly alter the combined dependent variables (multivariate F10,306 = 1.79, P = .06, η2 = .06, 1-ß = 0.83). While head to head, body to head, and stick to head mechanisms are penalty-inducing offenses in men's lacrosse, head to ground, head to ball, and combination impacts have similar head accelerations. If penalties and rules are created to protect players from traumatic head injury, the authors recommend stricter enforcement.

Reversal of high fat diet-induced obesity improves glucose tolerance, inflammatory response, ß-amyloid accumulation and cognitive decline in the APP/PSEN1 mouse model of Alzheimer's disease.

This study assessed the extent to which high fat diet (HFD)-induced ß-amyloid accumulation and cognitive decline in APP/PSEN1 mice are reversible through control of fat intake. Ten months of HFD (60% calories from fat) led to significant deficits in a 2-trial Y maze task, and nest building assay, and decreased voluntary locomotor activity. The HFD induced an inflammatory response, indicated by increased expression of several inflammatory markers. Substituting a low fat diet led to pronounced weight loss and correction of glucose intolerance, decreases in the inflammatory response, and improved performance on behavioral tasks in both wild-type and APP/PSEN1 transgenic mice. Insoluble ß-amyloid levels, and extent of tau phosphorylation were also lower following dietary reversal in APP/PSEN1 mice compared to high fat-fed animals, indicating that the inflammatory response may have contributed to key pathogenic pathways in the Alzheimer's disease model. The data suggest that weight loss can be a vital strategy for cognitive protection, but also highlight potential mechanisms for intervention when sustained weight loss is not possible.

Macrophage IKKα Deficiency Suppresses Akt Phosphorylation, Reduces Cell Survival, and Decreases Early Atherosclerosis.

OBJECTIVE: The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear. APPROACH AND RESULTS: Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S(473) phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα(-/-) monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)(-/-) mice reconstituted with IKKα(-/-) hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR(-/-) mice transplanted with wild-type cells. CONCLUSIONS: Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.

Jnk1 Deficiency in Hematopoietic Cells Suppresses Macrophage Apoptosis and Increases Atherosclerosis in Low-Density Lipoprotein Receptor Null Mice.

OBJECTIVE: The c-Jun NH2-terminal kinases (JNK) are regulated by a wide variety of cellular stresses and have been implicated in apoptotic signaling. Macrophages express 2 JNK isoforms, JNK1 and JNK2, which may have different effects on cell survival and atherosclerosis. APPROACH AND RESULTS: To dissect the effect of macrophage JNK1 and JNK2 on early atherosclerosis, Ldlr(-/-) mice were reconstituted with wild-type, Jnk1(-/-), and Jnk2(-/-) hematopoietic cells and fed a high cholesterol diet. Jnk1(-/-)→Ldlr(-/-) mice have larger atherosclerotic lesions with more macrophages and fewer apoptotic cells than mice transplanted with wild-type or Jnk2(-/-) cells. Moreover, genetic ablation of JNK to a single allele (Jnk1(+/-)/Jnk2(-/-) or Jnk1(-/-)/Jnk2(+/-)) in marrow of Ldlr(-/-) recipients further increased atherosclerosis compared with Jnk1(-/-)→Ldlr(-/-) and wild-type→Ldlr(-/-) mice. In mouse macrophages, anisomycin-mediated JNK signaling antagonized Akt activity, and loss of Jnk1 gene obliterated this effect. Similarly, pharmacological inhibition of JNK1, but not JNK2, markedly reduced the antagonizing effect of JNK on Akt activity. Prolonged JNK signaling in the setting of endoplasmic reticulum stress gradually extinguished Akt and Bad activity in wild-type cells with markedly less effects in Jnk1(-/-) macrophages, which were also more resistant to apoptosis. Consequently, anisomycin increased and JNK1 inhibitors suppressed endoplasmic reticulum stress-mediated apoptosis in macrophages. We also found that genetic and pharmacological inhibition of phosphatase and tensin homolog abolished the JNK-mediated effects on Akt activity, indicating that phosphatase and tensin homolog mediates crosstalk between these pathways. CONCLUSIONS: Loss of Jnk1, but not Jnk2, in macrophages protects them from apoptosis, increasing cell survival, and this accelerates early atherosclerosis.

Patient Outcomes Utilizing the Mulligan Concept of Mobilization With Movement to Treat Intercollegiate Patients Diagnosed With Lateral Ankle Sprain: An a Priori Case Series.

CONTEXT: While the incidence and reinjury rates of lateral ankle sprain (LAS) continue to persist at high rates across many sporting activities, further exploration of assessment and treatment beyond the traditional ligamentous and strength/proprioceptive model is warranted. Further, assessing and treating both arthrokinematic and osteokinematic changes associated with LAS can provide insight into a more diverse approach to treating ankle pathology. OBJECTIVE: To examine the clinical use of the Mulligan Concept mobilization with movement (MWM) while treating patients diagnosed with an acute grade I or II LAS through authentic patient care. DESIGN: An a priori case series. SETTING: Intercollegiate athletic training clinic. PATIENTS: Intercollegiate patients diagnosed with an acute grade I or II LAS. INTERVENTION: The Mulligan Concept distal fibular anterior to posterior MWM. MAIN OUTCOME MEASURES: Pain-Intensity Numeric Rating Scale (NRS) with Non-Weight Bearing (NRS-NWB) and Weight Bearing (NRS-WB), Disablement of the Physically Active Scale (DPAscale), Foot and Ankle Ability Measure (FAAM) with Activity of Daily living (FAMM-ADL) and Sport (FAAM-Sport), Client Specific Impairment Measure (CSIM), Y-Balance Composite (YBC), and Weight Bearing Measure for Dorsiflexion (WBDF). RESULTS: Patients who are diagnosed with an acute grade I or II LAS and are treated with the Mulligan Concept report immediate and long-lasting minimal clinically important differences in patient outcome measures. CONCLUSION: Clinicians who examine and use the Mulligan Concept MWM to treat acute LAS can expect immediate positive results that are progressively retained over time specific to patient-centered outcome measures as well as functional clinicianbased measures. Based on the immediate and positive results, clinicians should examine associated osteokinematic and arthrokinematic changes beyond that of the traditional ligamentous model.

Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton.

Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species.

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism.

Ascorbic acid prevents VEGF-induced increases in endothelial barrier permeability.

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 µM and complete inhibition at 50 µM. Loading cells with 100 µM ascorbate also decreased the basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25%, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 µM L-NAME (but not D-NAME) as well as by 30 µM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema.

Ascorbic acid transport in brain microvascular pericytes.

Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 min and an apparent transport Km of 21 µM, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion.

Sodium-dependent vitamin C transporter-2 mediates vitamin C transport at the cortical nerve terminal.

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice.

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr(-/-) mice reconstituted with Akt1(-/-) fetal liver cells (Akt1(-/-)→Ldlr(-/-)) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr(-/-)). In contrast, Akt2(-/-)→Ldlr(-/-) mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr(-/-) mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2(-/-)→Ldlr(-/-) mice had smaller aortic lesions compared with WT→Ldlr(-/-) and Akt1(-/-)→Ldlr(-/-) mice. Importantly, Akt2(-/-)→Ldlr(-/-) mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6C(hi) and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2(-/-) mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.

Ascorbate reverses high glucose- and RAGE-induced leak of the endothelial permeability barrier.

High glucose concentrations due to diabetes increase leakage of plasma constituents across the endothelial permeability barrier. We sought to determine whether vitamin C, or ascorbic acid (ascorbate), could reverse such high glucose-induced increases in endothelial barrier permeability. Human umbilical vein endothelial cells and two brain endothelial cell lines cultured at 25 mM glucose showed increases in endothelial barrier permeability to radiolabeled inulin compared to cells cultured at 5mM glucose. Acute loading of the cells for 30-60 min with ascorbate before the permeability assay prevented the high glucose-induced increase in permeability and decreased basal permeability at 5mM glucose. High glucose-induced barrier leakage was mediated largely by activation of the receptor for advanced glycation end products (RAGE), since it was prevented by RAGE blockade and mimicked by RAGE ligands. Intracellular ascorbate completely prevented RAGE ligand-induced increases in barrier permeability. The high glucose-induced increase in endothelial barrier permeability was also acutely decreased by several cell-penetrant antioxidants, suggesting that at least part of the ascorbate effect could be due to its ability to act as an antioxidant.

Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes.

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 µM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.