ALBERT

Description: The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions.Malasseziaare the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn’s disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within theMalasseziagenus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation inMalassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure ofMalasseziaflavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent ofMalasseziaflavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

Description: Recent studies have suggested that binding of oxygen to hemoglobin (Hb) facilitates reactions of nitric oxide (NO) that lead to production of S-nitrosohemoglobin (SNO-Hb), and that vasodilator S-nitrosthiol (SNO) is dispensed by red blood cells (RBCs) at low oxygen tension (pO2) to dilate blood vessels. In human lungs, NO bioactivity serves to attenuate hypoxic pulmonary vasoconstriction (HPV). We therefore considered the possibility that RBC-SNO may oppose HPV and that defective vasodilation by RBCs may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). Here we report that RBCs from patients with PAH exhibit substantial depletion of SNO-Hb and consequent impairment in hypoxia-mediated vasodilation. Furthermore, levels of RBC-NO correlated inversely with pulmonary artery pressures. A SNO-Hb deficiency characteristic of PAH was reproduced in control RBCs by hypoxia: loss of SNO-Hb was accompanied by a buildup of heme-NO species that are deficient in the pO2-governed intramolecular transfer of NO to cysteine thiol, yielding RBCs deficient in NO bioactivity. SNO-deficient RBCs produced exaggerated HPV responses as compared to SNO-replete RBCs. In PAH patients, SNO-Hb repletion fully restored the hypoxic vasodilator activity of RBCs. Our results suggest that a deficiency in RBC-SNO contributes to pulmonary hypertension and hypoxemia, and that repletion of RBC-SNO represents a rational strategy for treating PAH patients.

Description: Physiological O2 gradients are principal regulators of blood flow in the microcirculation: position-to-position changes in hemoglobin (Hb) O2 saturation are coupled to regulated vasodilation (“hypoxic vasodilation”). The mechanism by which graded changes in O2 content of blood evoke this response has been a great challenge to understand. A new role for red blood cells (RBCs) in hypoxic dilation of blood vessels and inhibition of platelet activation involving release of nitric oxide (NO) bioactivity is described. We show that NO groups can be transferred within hemoglobin (Hb) from hemes to highly-conserved cysteine thiols (β-Cys93) to form bioactive S-nitrosohemoglobin (SNO-Hb), and that efficient production of SNO-Hb requires selective processing of NO within the β-subunits. Bioactive SNO-Hb is localized primarily to the RBC membrane through interaction with Band 3, the transmembrane anion-exchanger 1 protein (AE1). Upon deoxygenation, transfer of the NO group from β-Cys93 of Hb to a cysteine thiol within AE1 serves the RBC vasodilator activity. In this way, O2 binding in Hb modulates the release of NO bioactivity. We further show that RBC NO bioactivity is inversely proportional to pO2 and impaired in disease. In an aortic ring bioassay sparged with variable concentrations of O2, addition of normal human RBCs elicited graded responses from relaxation at tissue pO2 (~3–7 mm Hg, hypoxic vasodilation), to loss of relaxation and progressively greater contractions at pO2’s of 10–63 mm Hg (hyperoxic vasoconstriction). Notably, RBC SNO-Hb levels and hypoxic vasodilation are impaired in several diseases characterized by vascular dysfunction. For example, in RBCs from patients with pulmonary arterial hypertension (PAH), we found decreased (13% of control) SNO-Hb content (assessed by photolysis-chemiluminescence) and impaired O2-dependent vasodilation (bioassay). RBCs from patients with other ischemic disorders have also been examined: RBCs demonstrate a pathogenesis-based impairment in their ability to mediate hypoxic vasodilation by NO. These results confirm the (patho)physiologic importance of RBC NO, and suggest that RBC dysfunction may contribute to impaired blood flow in diseases of the heart, lung and blood.

Description: Abstract 3251 Sickle erythrocytes (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, are deficient in nitric oxide (NO) content, and are inefficient in inducing vasodilation in response to hypoxia, all of which promote vascular occlusion, the hallmark of sickle cell disease (SCD). Furthermore, normal RBCs stored for transfusion are frequently used in SCD but are also NO-deficient. We therefore tested the hypothesis that repletion of intracellular SS or normal RBC NO could improve the abnormal circulatory characteristics of SSRBCs, using previously described in vitro (Zennadi et al. 2004 & 2008) and in vivo systems (Zennadi et al. 2007, Zhu et al. 2011). While exposure of SSRBCs to epinephrine (epi) up-regulated SSRBC adhesion to cultured HUVECs by 2.7±0.2-fold over baseline (p=0.0019), as previously described, loading SSRBCs with NO under hypoxic conditions (favoring SNO-Hb formation and bioactive NO availability), followed by reoxygenation prior to epi treatment, significantly decreased epi-induced SSRBC adhesion by 89±6.4% (p

Description: Introduction Nitric oxide (NO) is a vasoactive molecule that can bind to hemoglobin (Hb) in the form of S-nitrosothiol (SNO) functionalities at the β93 cysteine residues. Red blood cells (RBCs) containing S-nitrosohemoglobin (SNO-Hb) are able to not only deliver oxygen, but release vasodilatory NO/SNO equivalents to enhance blood flow in order to match tissue oxygen demand (e.g., during hypoxic vasodilation). Newborn babies normally carry two variants of hemoglobin, adult (Hb A) and fetal (Hb F). It is well known that Hb F binds oxygen more tightly than Hb A in order to facilitate oxygen scavenging across the placenta from maternal Hb A. Given that SNO-Hb is preferentially formed on oxygenated Hb, we hypothesized that Hb F may also bind a higher concentration of NO. Previous studies examining the NO content of cord blood were disadvantaged by looking at cord blood Hb as a whole. To date, no attempt has been made to determine the basal levels of NO bound to each Hb variant independently. Therefore, we aimed to separate the variants and measure the NO/SNO content of Hbs F and A in order to establish basal levels for each variant in cord blood at term. We reasoned that the results could improve insight into mechanisms of abnormal perinatal transitions and the selection of therapies involving NO signaling and/or RBC transfusion. Methods Venous and arterial umbilical cord blood samples were collected immediately after normal term cesarean sections of infants with minimum gestational age of 37 weeks. RBC samples were washed in pH 7.4 phosphate buffered saline (PBS) with 100 µM diethylene triamine pentaacetic acid (DTPA) chelator to preserve SNOs, and hypotonically lysed. Total Hb was obtained through purification of the lysate through a Sephadex G-25 column. Partially purified Hb (200 µL) was loaded onto a HiTrap Q HP anionic exchange column (5 mL column volume with 34 µm bead size) and subjected to an increasing ionic strength gradient of 0–0.15M NaCl in pH 8.4 Tris buffer. Spectrophotometric analysis corroborated the complete separation of the variants. Isoelectric focusing on a Perkin Elmer Hemoglobin Resolve gel for 50 minutes at 1500 V and 10–15 °C was used in conjunction with an AFSC Hemopure control to identify the respective variants in each fraction. Each Hb variant was reconcentrated in pH 7.4 PBS with 100 µM DTPA via centrifugation through pre-rinsed 10 kDa MW cutoff centrifugal filters. The SNO/NO content of each variant was analyzed by photolysis-chemiluminescence of paired samples diluted to 100 μM Hb with/without 600 μM HgCl2. For samples with lower Hb concentration, a 6-fold molar excess of HgCl2 was also used. The Hg (mercury) acts to cleave NO bound to thiols (i.e., SNO) and is unreactive towards FeNO complexes; thus the difference in the paired sample peaks indicates the amount of SNO-Hb present in the sample. Results In arterial cord blood samples, the amount of SNO-Hb bound to each variant (i.e., fetal and adult) was found to average ∼5 x 10-4 mol SNO per mol of Hb tetramer (Figure 1A). There was no significant difference between Hb F and Hb A with regards to SNO-Hb. Venous cord blood samples had similar results. The amount of total NO (i.e., SNO-Hb and heme-bound NO) bound to each variant was significantly higher on Hb A in arterial samples. (Figure 1B, *, p value 〈 0.05 vs. Hb F by paired t-test). Conclusions Both Hb A and Hb F carry substantial and similar amounts of SNO adduct. Given the lower percentage (∼15-20%) of Hb A as a constituent in the total Hb of cord blood at term, the finding of a higher total NO content on Hb A than Hb F suggests that the presence of Hb A may be important to the total NO bioavailability in newborns. Vasodilator NO/SNO is known to be essential in the healthy cardiopulmonary transition to air breathing at birth. Thus, fundamental and translational studies assessing these species in newborns experiencing difficult transitions or pathophysiological states (e.g., persistent pulmonary hypertension of the newborn, PPHN) may provide potential biomarkers with utility in early detection of disease, prognosis, and intervention selection and management. The results and methodology presented here have broad applications to numerous areas of hematology and other medicine including neonatal intensive care, therapeutic induction of HbF in sickle cell disease patients, and decision-making in transfusion medicine. Disclosures: No relevant conflicts of interest to declare.

Description: Transfusion of red blood cells (RBCs) is associated with excess mortality and morbidity in some anemic patients with critical illness, and no benefit in others, but the underlying mechanisms are poorly understood. Specifically, little is known of how “storage lesions” of the RBC may contribute to adverse outcomes or lack of benefit from a transfusion. The recent discovery of an active role of the human RBC in regulating blood flow – a major determinant of its own function, O2 delivery – prompts a reexamination of storage-induced changes in this and related RBC functions, and motivates specific identification of the relevant mediators. RBCs release the vasodilators S-nitrosothiols (SNOs) and ATP upon physiological exposure to hypoxia, contributing to O2-dependent blood flow regulation. The result is hypoxic vasodilation peripherally and modulation of hypoxic vasoconstriction in the lung. We show that conventionally stored human RBCs become deficient in SNOs within three hours of acquisition and independent of exposure to storage solution or leukofiltration; both hemoglobin-bound SNO and membrane SNO are depressed. Early changes (hours) are also seen in the function of stored RBCs, namely in RBC regulation of vascular tone in vitro and in the lungs of intact mice in vivo, whereas declines in RBC deformability take place more gradually (from days to weeks). We also confirm storage-induced depletion of RBC ATP, a lesion which may contribute to the functional declines in both deformability and in RBC-dependent vasoactivity. Time courses, therefore, vary widely for several storage-induced functional and biochemical defects that may contribute to adverse clinical outcomes following RBC transfusion. Strategies that either prevent the loss of vasoactive mediators during RBC storage, or replete bioactive (S)NO after RBC storage, are reviewed and may inform the rational design of strategies to improve the risk-benefit balance associated with RBC transfusion in critically ill and other anemic patients.