Jaideep Honavar

The apolipoprotein A-I mimetic peptide 4F prevents defects in vascular function in endotoxemic rats

High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce inflammatory responses to lipopolysaccharide (LPS). We tested the hypothesis that the apoA-I mimetic peptide 4F prevents LPS-induced defects in blood pressure and vascular reactivity. Systolic blood pressure (SBP) was measured in rats at baseline and 6 h after injection of LPS (10 mg/kg) or saline vehicle. Subgroups of LPS-treated rats also received 4F (10 mg/kg) or scrambled 4F (Sc-4F). LPS administration reduced SBP by 35% compared with baseline. 4F attenuated the reduction in SBP in LPS-treated rats (17% reduction), while Sc-4F was without effect. Ex vivo studies showed a reduced contractile response to phenylephrine (PE) in aortae of LPS-treated rats (ED50 = 459 ± 83 nM) compared with controls (ED50 = 57 ± 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED50 = 60 ± 9 nM), reduced aortic NOS2 protein, normalized plasma levels of NO metabolites, and reduced mortality in LPS-treated rats. These changes were associated with a reduction in plasma endotoxin activity. In vivo administration of 14C-4F and Bodipy-LPS resulted in their colocalization and retention in the HDL fraction. It is proposed that 4F promotes the localization of LPS to the HDL fraction, resulting in endotoxin neutralization. 4F may thus prevent LPS-induced hemodynamic changes associated with NOS2 induction.

Chlorine Gas Exposure Causes Systemic Endothelial Dysfunction by Inhibiting Endothelial Nitric Oxide Synthase–Dependent Signaling

Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

Anti-Inflammatory Mechanisms of Apolipoprotein A-I Mimetic Peptide in Acute Respiratory Distress Syndrome Secondary to Sepsis

Acute respiratory distress syndrome (ARDS) due to sepsis has a high mortality rate with limited treatment options. High density lipoprotein (HDL) exerts innate protective effects in systemic inflammation. However, its role in ARDS has not been well studied. Peptides such as L-4F mimic the secondary structural features and functions of apolipoprotein (apo)A-I, the major protein component of HDL. We set out to measure changes in HDL in sepsis-mediated ARDS patients, and to study the potential of L-4F to prevent sepsis-mediated ARDS in a rodent model of lipopolysaccharide (LPS)-mediated acute lung injury, and a combination of primary human leukocytes and human ARDS serum. We also analyzed serum from non-lung disease intubated patients (controls) and sepsis-mediated ARDS patients. Compared to controls, ARDS demonstrates increased serum endotoxin and IL-6 levels, and decreased HDL, apoA-I and activity of anti-oxidant HDL-associated paraoxanase-1. L-4F inhibits the activation of isolated human leukocytes and neutrophils by ARDS serum and LPS in vitro. Further, L-4F decreased endotoxin activity and preserved anti-oxidant properties of HDL both in vitro and in vivo. In a rat model of severe endotoxemia, L-4F significantly decreased mortality and reduces lung and liver injury, even when administered 1 hour post LPS. Our study suggests the protective role of the apoA-I mimetic peptide L-4F in ARDS and gram-negative endotoxemia and warrant further clinical evaluation. The main protective mechanisms of L-4F are due to direct inhibition of endotoxin activity and preservation of HDL anti-oxidant activity.

Administration of nitrite after chlorine gas exposure prevents lung injury: effect of administration modality.

Cl(2) gas toxicity is complex and occurs during and after exposure, leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl(2) exposure can occur in diverse situations encompassing mass casualty scenarios, highlighting the need for postexposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we assessed the efficacy of a single dose of nitrite (1 mg/kg) to decrease ALI when administered to rats via intraperitoneal (ip) or intramuscular (im) injection 30 min after Cl(2) exposure. Exposure of rats to Cl(2) gas (400 ppm, 30 min) significantly increased ALI and caused RAS 6-24h postexposure as indexed by BAL sampling of lung surface protein and polymorphonucleocytes (PMNs) and increased airway resistance and elastance before and after methacholine challenge. Intraperitoneal nitrite decreased Cl(2)-dependent increases in BAL protein but not PMNs. In contrast im nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase-dependent manner. Histological evaluation of airways 6h postexposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl(2)-exposed rats. Both ip and im nitrite improved airway histology compared to Cl(2) gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with im compared to ip nitrite. Airways were rendered more sensitive to methacholine-induced resistance and elastance after Cl(2) gas exposure. Interestingly, im nitrite, but not ip nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of im and ip therapy showed a twofold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl(2) exposure-dependent increases in circulating leukocytes. Halving the im nitrite dose resulted in no effect in PMN accumulation but significant reduction of BAL protein levels, indicating a distinct nitrite dose dependence for inhibition of Cl(2)-dependent lung permeability and inflammation. These data highlight the potential for nitrite as a postexposure therapeutic for Cl(2) gas-induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.

Red blood cell washing, nitrite therapy, and antiheme therapies prevent stored red blood cell toxicity after trauma–hemorrhage

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma-hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0-10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.

Effects of Erythrocyte Aging on Nitric Oxide and Nitrite Metabolism

AIMS Recent studies have suggested that in addition to oxygen transport, red blood cells (RBC) are key regulators of vascular function by both inhibiting and promoting nitric oxide (NO)-mediated vasodilation. Most studies assume that RBC are homogenous, but, in fact, they comprise cells of differing morphology and biochemical composition which are dependent on their age, parameters that control NO reactions. We tested the hypothesis that distinct RBC populations will have differential effects on NO signaling. RESULTS Young and old RBC were separated by density gradient centrifugation. Consistent with previous reports, old RBC had decreased levels of surface N-acetyl neuraminic acid and increased oxygen binding affinities. Competition kinetic experiments showed that older RBCs scavenged NO∼2-fold faster compared with younger RBC, which translated to a more potent inhibition of both acetylcholine and NO-donor dependent vasodilation of isolated aortic rings. Moreover, nitrite oxidation kinetics was faster with older RBC compared with younger RBC; whereas no differences in nitrite-reduction kinetics were observed. This translated to increased inhibitory effect of older RBC to nitrite-dependent vasodilation under oxygenated and deoxygenated conditions. Finally, leukodepleted RBC storage also resulted in more dense RBC, which may contribute to the greater NO-inhibitory potential of stored RBC. INNOVATION These results suggest that a key element in vascular NO-homeostasis mechanisms is the distribution of RBC ages across the physiological spectrum (0-120 days) and suggest a novel mechanism for inhibited NO bioavailability in diseases which are characterized by a shift to an older RBC phenotype. CONCLUSION Older RBC inhibit NO bioavailability by increasing NO- and nitrite scavenging.

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)‐based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome‐encapsulated Hb (PEG‐LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG‐LEH dispersions using stopped‐flow UV–visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG‐LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG‐LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO‐mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG‐LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.

Formation of chlorinated lipids post-chlorine gas exposure

Exposure to chlorine (Cl2) gas can occur during accidents and intentional release scenarios. However, biomarkers that specifically indicate Cl2 exposure and Cl2-derived products that mediate postexposure toxicity remain unclear. We hypothesized that chlorinated lipids (Cl-lipids) formed by direct reactions between Cl2 gas and plasmalogens serve as both biomarkers and mediators of post-Cl2 gas exposure toxicities. The 2-chloropalmitaldehyde (2-Cl-Pald), 2-chlorostearaldehyde (2-Cl-Sald), and their oxidized products, free- and esterified 2-chloropalmitic acid (2-Cl-PA) and 2-chlorostearic acid were detected in the lungs and plasma of mouse and rat models of Cl2 gas exposure. Levels of Cl-lipids were highest immediately post-Cl2 gas exposure, and then declined over 72 h with levels remaining 20- to 30-fold higher at 24 h compared with baseline. Glutathione adducts of 2-Cl-Pald and 2-Cl-Sald also increased with levels peaking at 4 h in plasma. Notably, 3-chlorotyrosine also increased after Cl2 gas exposure, but returned to baseline within 24 h. Intranasal administration of 2-Cl-PA or 2-Cl-Pald at doses similar to those formed in the lung after Cl2 gas exposure led to increased distal lung permeability and inflammation and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation. These data suggest that Cl-lipids could serve as biomarkers and mediators for Cl2 gas exposure and toxicity.

Chlorine gas exposure disrupts nitric oxide homeostasis in the pulmonary vasculature

Exposure to chlorine (Cl2) gas during industrial accidents or chemical warfare leads to significant airway and distal lung epithelial injury that continues post exposure. While lung epithelial injury is prevalent, relatively little is known about whether Cl2 gas also promotes injury to the pulmonary vasculature. To determine this, rats were subjected to a sub-lethal Cl2 gas exposure (400 ppm, 30 min) and then brought back to room air. Pulmonary arteries (PA) were isolated from rats at various times post-exposure and contractile (phenylephrine) and nitric oxide (NO)-dependent vasodilation (acetylcholine and mahmanonoate) responses measured ex vivo. PA contractility did not change, however significant inhibition of NO-dependent vasodilation was observed that was maximal at 24-48 h post exposure. Superoxide dismutase restored NO-dependent vasodilation suggesting a role for increased superoxide formation. This was supported by ∼2-fold increase in superoxide formation (measured using 2-hydroethidine oxidation to 2-OH-E+) from PA isolated from Cl2 exposed rats. We next measured PA pressures in anesthetized rats. Surprisingly, PA pressures were significantly (∼4 mmHg) lower in rats that had been exposed to Cl2 gas 24 h earlier suggesting that deficit in NO-signaling observed in isolated PA experiments did not manifest as increased PA pressures in vivo. Administration of the iNOS selective inhibitor 1400W, restored PA pressures to normal in Cl2 exposed, but not control rats suggesting that any deficit in NO-signaling due to increased superoxide formation in the PA, is offset by increased NO-formation from iNOS. These data indicate that disruption of endogenous NO-signaling mechanisms that maintain PA tone is an important aspect of post-Cl2 gas exposure toxicity.

Nitrite therapy improves survival postexposure to chlorine gas.

Exposure to relatively high levels of chlorine (Cl₂) gas can occur in mass-casualty scenarios associated with accidental or intentional release. Recent studies have shown a significant postexposure injury phase to the airways, pulmonary, and systemic vasculatures mediated in part by oxidative stress, inflammation, and dysfunction in endogenous nitric oxide homeostasis pathways. However, there is a need for therapeutics that are amenable to rapid and easy administration in the field and that display efficacy toward toxicity after chlorine exposure. In this study, we tested whether nitric oxide repletion using nitrite, by intramuscular injection after Cl₂ exposure, could prevent Cl₂ gas toxicity. C57bl/6 male mice were exposed to 600 parts per million Cl₂ gas for 45 min, and 24-h survival was determined with or without postexposure intramuscular nitrite injection. A single injection of nitrite (10 mg/kg) administered either 30 or 60 min postexposure significantly improved 24-h survival (from ∼20% to 50%). Survival was associated with decreased neutrophil accumulation in the airways. Rendering mice neutropenic before Cl₂ exposure improved survival and resulted in loss of nitrite-dependent survival protection. Interestingly, female mice were more sensitive to Cl₂-induced toxicity compared with males and were also less responsive to postexposure nitrite therapy. These data provide evidence for efficacy and define therapeutic parameters for a single intramuscular injection of nitrite as a therapeutic after Cl₂ gas exposure that is amenable to administration in mass-casualty scenarios.