Bernard J. Fisher

Mechanisms of attenuation of abdominal sepsis induced acute lung injury by ascorbic acid.

Bacterial infections of the lungs and abdomen are among the most common causes of sepsis. Abdominal peritonitis often results in acute lung injury (ALI). Recent reports demonstrate a potential benefit of parenteral vitamin C [ascorbic acid (AscA)] in the pathogenesis of sepsis. Therefore we examined the mechanisms of vitamin C supplementation in the setting of abdominal peritonitis-mediated ALI. We hypothesized that vitamin C supplementation would protect lungs by restoring alveolar epithelial barrier integrity and preventing sepsis-associated coagulopathy. Male C57BL/6 mice were intraperitoneally injected with a fecal stem solution to induce abdominal peritonitis (FIP) 30 min prior to receiving either AscA (200 mg/kg) or dehydroascorbic acid (200 mg/kg). Variables examined included survival, extent of ALI, pulmonary inflammatory markers (myeloperoxidase, chemokines), bronchoalveolar epithelial permeability, alveolar fluid clearance, epithelial ion channel, and pump expression (aquaporin 5, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and Na(+)-K(+)-ATPase), tight junction protein expression (claudins, occludins, zona occludens), cytoskeletal rearrangements (F-actin polymerization), and coagulation parameters (thromboelastography, pro- and anticoagulants, fibrinolysis mediators) of septic blood. FIP-mediated ALI was characterized by compromised lung epithelial permeability, reduced alveolar fluid clearance, pulmonary inflammation and neutrophil sequestration, coagulation abnormalities, and increased mortality. Parenteral vitamin C infusion protected mice from the deleterious consequences of sepsis by multiple mechanisms, including attenuation of the proinflammatory response, enhancement of epithelial barrier function, increasing alveolar fluid clearance, and prevention of sepsis-associated coagulation abnormalities. Parenteral vitamin C may potentially have a role in the management of sepsis and ALI associated with sepsis.

Prolyl hydroxylase inhibition attenuates post-ischemic cardiac injury via induction of endoplasmic reticulum stress genes.

Ischemia/reperfusion (I/R) unleashes cellular events that threaten organ survival. I/R affects endoplasmic reticulum (ER) integrity and initiates the unfolded protein response (UPR). The adaptive arm of the UPR attenuates ER stress by increasing expression of chaperones promoting proper protein folding. However, failure to resolve ER stress leads to apoptotis. We recently showed that prolyl hydroxylase inhibition (PHI) attenuated post-ischemic cardiac injury. We hypothesized that PHI attenuated myocardial I/R injury through modulation of the UPR. We show for the first time that PHI activates all three regulatory arms of the UPR in murine microvascular endothelial cells and in mouse hearts. Cardiac I/R activated expression of pro-apoptotic CHOP (2.8 fold, n=3, p<0.01). PHI significantly decreased CHOP expression (50%, n=3, p<0.05) in post-ischemic hearts. PHI also induced activating transcription factor 4 (3.5 fold, n=3, p<0.001), glucose-regulated protein 78 (6 fold, n=3, p<0.001) and ER degradation-enhancing alpha-mannosidase-like protein (2.8 fold, n=3, p<0.001) expression in reperfusing hearts. Thus PHI resulted in significant reduction of apoptosis in post-ischemic myocardium. Our studies suggest that PHI induces protective ER stress proteins and attenuates post-ischemic myocardial damage by decreasing the pro-apoptotic components of the UPR.

Reoxygenating microvascular endothelium exhibits temporal dissociation of NF-κB and AP-1 activation

Alterations of cellular redox balance in microvascular endothelium results in changes of essential cell functions. These alterations may arise, in part, due to modifications in the pattern of gene expression produced by transcription factor activation. Endothelium subjected to hypoxia/reoxygenation becomes redox imbalanced, thereby leading to activation and perhaps production of a proinflammatory state. A human dermal microvascular endothelial cell line (HMEC-1) was exposed to 6 h of hypoxia (3% O(2)) followed by return to normoxia atmospheric conditions. Reactive oxygen species (ROS) generation (dichlorofluoroscein epifluorescence) was immediate and significant following reoxygenation. Electrophoretic mobility shift assays revealed activation of the oxidant sensitive transcription factors NFkappaB and AP-1, though importantly, peak activation of each factor was separated temporally by greater than 60 min. NFkappaB activation occurred without degradation of the inhibitory protein IkappaBalpha. Reoxygenating HMEC-1 exhibited a greater than 500-fold increase in polymorphonuclear neutrophil (PMN) adhesion when compared to normoxic controls. Exposure of reoxygenating HMEC-1 to the antioxidant pyrrolidine dithiocarbamate produced complete abrogation of NFkappaB activation and the intensive PMN adhesion observed in untreated, posthypoxic HMEC-1. Though rexoygenation stress induced significant upregulation of PMN adhesion, no upregulation of interleukin-8 production was observed. Our results suggest that ROS generation occurring in endothelium following onset of reoxygenation stress signals activation of key transcription factors and that their activation takes place in a temporal fashion. The temporal feature of transcription factor activation may be key to production of a postischemic proinflammatory state.

Attenuation of Sepsis-induced Organ Injury in Mice by Vitamin C

BACKGROUND Multiple organ dysfunction syndrome (MODS) is the principal cause of death in patients with sepsis. Recent work supports the notion that parenteral vitamin C (VitC) is protective in sepsis through pleiotropic mechanisms. Whether suboptimal levels of circulating VitC increase susceptibility to sepsis-induced MODS is unknown. MATERIALS AND METHODS Unlike mice, humans lack the ability to synthesize VitC because of loss of L-gulono-γ-lactone oxidase (Gulo), the final enzyme in the biosynthesis of VitC. To examine whether physiological levels of VitC are required for defense against a catastrophic infection, we induced sepsis in VitC sufficient and VitC deficient Gulo(-/-) mice by intraperitoneal infusion of a fecal stem solution (FIP). Some VitC deficient Gulo(-/-) mice received a parenteral infusion of ascorbic acid (AscA, 200 mg/kg) 30 minutes after induction of FIP. We used molecular, histological, and biochemical analyses to assess for MODS as well as abnormalities in the coagulation system and circulating blood cells. RESULTS FIP produced injury to lungs, kidneys and liver (MODS) in VitC deficient Gulo(-/-) mice. MODS was not evident in FIP-exposed VitC sufficient Gulo(-/-) mice and attenuated in VitC deficient Gulo(-/-) mice infused with AscA. Septic VitC deficient Gulo(-/-) mice developed significant abnormalities in the coagulation system and circulating blood cells. These were attenuated by VitC sufficiency/infusion in septic Gulo(-/-) mice. CONCLUSIONS VitC deficient Gulo(-/-) mice were more susceptible to sepsis-induced MODS. VitC sufficiency or parenteral infusion of VitC, following induction of sepsis, normalized physiological functions that attenuated the development of MODS in sepsis.

Atypical mechanism of NF-κB activation during reoxygenation stress in microvascular endothelium: a role for tyrosine kinases

The transcription factor nuclear factor kappaB (NF-kappaB) regulates genes that contribute to acute inflammatory reactions in cytokine-activated endothelium. Tumor necrosis factor activates NF-kappaB through serine phosphorylation, induced by inhibitor kappaB kinases (IKK), and subsequent degradation of inhibitor kappaB (IkappaB). In contrast to cytokine stress, our studies show that oxidative stress, generated by exposure to hypoxia followed by reoxygenation (H/R), failed to activate IKK in human microvascular endothelial cells (HMEC-1). We report an alternative mechanism for NF-kappaB activation during H/R stress without IkappaBalpha degradation. This mechanism involves activation of protein tyrosine kinases (PTK) that phosphorylate IkappaBalpha with peak phosphorylation occurring after 30 min of reoxygenation. Involvement of PTK was reinforced by the demonstration that the PTK inhibitor, herbimycin A, prevented H/R-mediated NF-kappaB activation. Tyrosine phosphorylation alters the association between IkappaBalpha and NF-kappaB with sufficient intensity to allow transient NF-kappaB translocation to the cell nuclei within 45 min of onset of reoxygenation stress. Immunofluorescence imaging of NF-kappaB protein reveals it to be shuttled between the nucleus and cytoplasm within 90 min of reoxygenation. Furthermore, IkappaBalpha appears to be associated with NF-kappaB during the nucleo-cytoplasmic shuttling and is thus protected from degradation. Overall, these studies suggest that tyrosine phosphorylation of IkappaBalpha represents a proteolysis-independent mechanism of NF-kappaB activation that can be targeted for preventing H/R-mediated injury without affecting normal inflammatory responses.

Regulation of hypoxia inducible factor-1 by nitric oxide in contrast to hypoxia in microvascular endothelium.

Hypoxia activates the transcription factor, hypoxia inducible factor‐1 (HIF‐1). Besides hypoxia, HIF‐1 can be activated under normoxic conditions by nitric oxide. The signal transduction pathways involved in HIF‐1α stabilization, HIF‐1 DNA binding and transactivation by NO and hypoxia in microvascular endothelium remains unknown. We report that protein phosphorylation is involved in HIF‐1 activation during hypoxia and NO. The phosphatidylinositol 3‐kinase (PI‐3K)/Akt pathway has differential effects on HIF‐1 activation by hypoxia and NO. Our data indicate that the PI‐3K/Akt pathway is insufficient for HIF‐1α induction by hypoxia. The lipid and protein phosphatase activities of PTEN also appear to be involved in regulation of HIF‐1α by NO.

Vitamin C promotes wound healing through novel pleiotropic mechanisms

Vitamin C (VitC) or ascorbic acid (AscA), a cofactor for collagen synthesis and a primary antioxidant, is rapidly consumed post‐wounding. Parenteral VitC administration suppresses pro‐inflammatory responses while promoting anti‐inflammatory and pro‐resolution effects in human/murine sepsis. We hypothesised that VitC could promote wound healing by altering the inflammatory, proliferative and remodelling phases of wound healing. Mice unable to synthesise VitC (Gulo−/−) were used in this study. VitC was provided in the water (sufficient), withheld from another group (deficient) and supplemented by daily intra‐peritoneal infusion (200 mg/kg, deficient + AscA) in a third group. Full thickness excisional wounds (6 mm) were created and tissue collected on days 7 and 14 for histology, quantitative polymerase chain reaction (qPCR) and Western blotting. Human neonatal dermal fibroblasts (HnDFs) were used to assess effects of In conclusion, VitC favorably on proliferation. Histological analysis showed improved wound matrix deposition and organisation in sufficient and deficient +AscA mice. Wounds from VitC sufficient and deficient + AscA mice had reduced expression of pro‐inflammatory mediators and higher expression of wound healing mediators. Supplementation of HnDF with AscA induced the expression of self‐renewal genes and promoted fibroblast proliferation. VitC favourably impacts the spatiotemporal expression of transcripts associated with early resolution of inflammation and tissue remodelling.

Impact of high dose vitamin C on platelet function

AIM To examine the effect of high doses of vitamin C (VitC) on ex vivo human platelets (PLTs). METHODS Platelet concentrates collected for therapeutic or prophylactic transfusions were exposed to: (1) normal saline (control); (2) 0.3 mmol/L VitC (Lo VitC); or (3) 3 mmol/L VitC (Hi VitC, final concentrations) and stored appropriately. The VitC additive was preservative-free buffered ascorbic acid in water, pH 5.5 to 7.0, adjusted with sodium bicarbonate and sodium hydroxide. The doses of VitC used here correspond to plasma VitC levels reported in recently completed clinical trials. Prior to supplementation, a baseline sample was collected for analysis. PLTs were sampled again on days 2, 5 and 8 and assayed for changes in PLT function by: Thromboelastography (TEG), for changes in viscoelastic properties; aggregometry, for PLT aggregation and adenosine triphosphate (ATP) secretion in response to collagen or adenosine diphosphate (ADP); and flow cytometry, for changes in expression of CD-31, CD41a, CD62p and CD63. In addition, PLT intracellular VitC content was measured using a fluorimetric assay for ascorbic acid and PLT poor plasma was used for plasma coagulation tests [prothrombin time (PT), partial thrombplastin time (PTT), functional fibrinogen] and Lipidomics analysis (UPLC ESI-MS/MS). RESULTS VitC supplementation significantly increased PLTs intracellular ascorbic acid levels from 1.2 mmol/L at baseline to 3.2 mmol/L (Lo VitC) and 15.7 mmol/L (Hi VitC, P < 0.05). VitC supplementation did not significantly change PT and PTT values, or functional fibrinogen levels over the 8 d exposure period (P > 0.05). PLT function assayed by TEG, aggregometry and flow cytometry was not significantly altered by Lo or Hi VitC for up to 5 d. However, PLTs exposed to 3 mmol/L VitC for 8 d demonstrated significantly increased R and K times by TEG and a decrease in the α-angle (P < 0.05). There was also a fall of 20 mm in maximum amplitude associated with the Hi VitC compared to both baseline and day 8 saline controls. Platelet aggregation studies, showed uniform declines in collagen and ADP-induced platelet aggregations over the 8-d study period in all three groups (P > 0.05). Collagen and ADP-induced ATP secretion was also not different between the three groups (P > 0.05). Finally, VitC at the higher dose (3 mmol/L) also induced the release of several eicosanoids including thromboxane B2 and prostaglandin E2, as well as products of arachidonic acid metabolism via the lipoxygenases pathway such as 11-/12-/15-hydroxyicosatetraenoic acid (P < 0.05). CONCLUSION Alterations in PLT function by exposure to 3 mmol/L VitC for 8 d suggest that caution should be exerted with prolonged use of intravenous high dose VitC.

Vitamin C Infusion for Gastric Acid Aspiration-Induced Acute Respiratory Distress Syndrome (ARDS)

1Division of Anesthesia Critical Care, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA 2Division of Pulmonary Disease and Critical Care Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA 3Division of Cardiothoracic Surgery, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA *Corresponding author Alpha A. Fowler, III, MD Professor of Medicine Division of Pulmonary Disease and Critical Care Medicine Virginia Commonwealth University Department of Internal Medicine Box 980050, Richmond VA 23298, USA Tel. 804-828-9071 Fax: 802-828-2578 E-mail: alpha.fowler@vcuhealth.org

Redox Potential Measurements in Red Blood Cell Packets Using Nanoporous Gold Electrodes

The redox potential of packed red blood cells (RBCs) was measured over a 56-day storage period using a newly developed potentiometric methodology consisting of a nanoporous gold electrode and a silver chloride coated silver reference electrode. Both milliliter- and microliter-sized volumes were separately evaluated. The addition of Vitamin C (VitC) in differing doses to the packed RBCs was also assessed as a means to improve redox stability and prolong storage duration. For RBCs containing only saline, the open-circuit potential (OCP) was ∼ -80 mV vs Ag/AgCl and drifted slightly with time; greater differences were also noted between different electrodes. The addition of exogenous VitC to the RBC shifts the OCP to more negative values, stabilizes the redox potential, and improves reproducibly between different electrodes due to the poising of blood. Over the 56-day storage period, the redox potential of the RBCs increased slightly, which can be attributed to change in pH and/or increasing oxidative stress during storage. Cyclic voltammograms acquired after open-circuit potential measurements showed a characteristic peak attributed to the oxidation of VitC. This peak decreased during storage with a time constant of 20.8 days. Likewise, the intercellular concentration of VitC increased with a time constant of 20.2 days as measured using a fluorescence assay. Collectively, these results demonstrate the usefulness of electrochemical measurements in the study of stored blood products.