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Biomedical subjects

Mark T Gladwin

Publications and source records attributed to Mark T Gladwin.

At least 19 recordsLinked to original sources
PubMed2026

Design, rationale, and baseline patient characteristics for the Sickle Cell Disease and CardiovAscular Risk-Red cell Exchange (SCD-CARRE) trial.

BACKGROUND: Despite wide utilization of automated red blood cell exchange (RBCX) transfusion in adult patients with sickle cell disease (SCD), no consensus or quality efficacy data exist on its use. The Sickle Cell Disease and CardiovAscular Risk- Red cell Exchange (SCD-CARRE) trial tests the hypothesis that an automated chronic RBCX transfusion strategy reduces acute health care encounters and death while improving quality of life and end-organ function (cardiac, pulmonary and renal) in participants with SCD that are at high risk of death. METHODS: Adult patients with SCD with elevated tricuspid regurgitant jet velocity (TRV) and/or chronic kidney disease were considered to be at high risk of death and were randomly assigned to RBCX plus standard of care vs standard of care alone. Participants assigned to RBCX received 12 months of exchange transfusions to maintain target pretransfusion hemoglobin S% < 30%, post-transfusion hemoglobin S% < 20%, and post-transfusion hemoglobin concentration &#x2265;10 g/dL. All study participants were managed according to NHLBI/ASH/ATS Expert Panel guidelines. The primary endpoint was the number of SCD acute health care encounters or death over 13 months. Secondary endpoints included measures of cardiovascular and renal function, exercise capacity, patient reported outcomes (all collected at baseline, and months 4, 8, and 12), and transfusion-related adverse events (collected monthly). RESULTS: Between 2020 and 2025, the SCD-CARRE trial randomized 173 participants at 23 sites across 3 countries. Enrolled participants had mean (SD) age of 45.8 (11.8) years and 54% were female. At baseline, participants had average TRV of 2.8 (0.5) m/s such that 45.9% had a TRV between 2.5 to 2.9 m/sec and 28.1% had a TRV &#x2265; 3.0 m/sec. The median (Q1, Q3) eGFR in this cohort was 60 (36, 110) mL/min/1.73 m2. The median (Q1, Q3) 6-minute walk test distance was 375 meters (309, 439), the median daily steps were 3,728 (2,187, 5,821), and participants experienced a median (Q1, Q3) of 2 (1, 5) pain episodes in the year prior to randomization. The trial results are pending. CONCLUSIONS: The SCD-CARRE trial successfully enrolled a cohort of n = 173 adults with SCD. This study highlights a rationale to evaluate the effect of automated chronic RBCX transfusion strategy plus standard of care as compared to standard of care alone in SCD patients at high risk of death with a focus on patient centered outcomes, preservation of cardiovascular function, end-organ complications and death. TRIAL REGISTRATION: ClinicalTrials.gov, Identifier: NCT04084080, https://clinicaltrials.gov/study/NCT04084080.

Adult↗
PubMed2003

Imaging hemoglobin oxygen saturation in sickle cell disease patients using noninvasive visible reflectance hyperspectral techniques: effects of nitric oxide.

Sickle cell disease is characterized by microvascular occlusion and hemolytic anemia, factors that impair tissue oxygen delivery. We use visible reflectance hyperspectral imaging to quantitate skin tissue hemoglobin oxygen saturation (HbO2) and to determine whether changes in blood flow during nitric oxide (NO) stimulation or gas administration (therapies proposed for this disease) improve skin tissue oxygen saturation in five patients with sickle cell disease. Compared with six healthy African-American subjects, sickle cell patients exhibited higher forearm blood flows (7.4 +/- 1.8 vs. 3.2 +/- 0.4 ml.min-1.100 ml tissue-1, P = 0.037) but significantly reduced percentages of skin HbO2 (61.0 +/- 0.2 vs. 77.5 +/- 0.2%, P < 0.001). Administration of acetylcholine to patients increased blood flow by 15.1 +/- 3.8 ml.min-1.100 ml tissue-1 and the percentage of skin HbO2 by 4.1 +/- 0.3% (P = 0.02, P < 0.001, respectively, from baseline values). Sodium nitroprusside, a direct NO donor, increased blood flow by 3.9 +/- 1.1 ml/min and the percentage of skin HbO2 by 2.9 +/- 0.3% (P = 0.02, P < 0.001, respectively). NO inhalation had no effect on forearm blood flow, yet increased the percentage of skin HbO2 by 2.3 +/- 0.3% (P < 0.001). Percentages of skin HbO2 were exponentially related to blood flow (R = 0.97, P < 0.001), indicating a limit to skin tissue oxygen saturation at high blood flows. Thus, for acetylcholine infusion leading to blood flows sevenfold greater than those of healthy resting African-American subjects, patients still exhibited lower percentages of skin HbO2 (65.2 +/- 0.2 vs. 77.5 +/- 0.2%, P < 0.001). Visible reflectance hyperspectral imaging demonstrates that either the stimulation or the administration of NO pharmacologically or by gas inhalation improves, but does not normalize, skin tissue oxygen saturation in patients with sickle cell disease.

Acetylcholine↗
PubMed2003

Interferon-gamma induces p11 gene and protein expression in human epithelial cells through interferon-gamma-activated sequences in the p11 promoter.

The effect of interferon (IFN)-gamma on p11 expression was studied in two human epithelial cell lines (BEAS-2B and HeLa). Treatment with IFN-gamma resulted in increased steady-state levels of p11 mRNA and protein expression, with a time-dependent and dose-dependent effect. Transient transfection experiments of a reporter gene construct containing 1498 bp of the 5'-flanking region of the p11 promoter demonstrated that IFN-gamma induced p11 gene expression at the transcriptional level. These effects were inhibited at the promoter and protein levels by a specific JAK-2 kinase inhibitor, AG-490. Functional analysis of the p11 promoter indicates that two gamma-activated sequence elements (GAS) located at positions 1219 and 1090 are important for the induction of the p11 promoter by IFN-gamma. Transfection of mutated reporter constructs demonstrated that the mutation at the GAS-2 site (1090) inhibited the p11 promoter activity, with a reduction of about approximately 73% and mutation at the GAS-3 site (1219) eliminated about 26% of the p11 promoter activity. A STAT1 dominant negative mutant vector at Tyr-701 (JAK kinase phosphorylation site) blocked the effect of IFN-gamma on the p11 promoter activity. IFN-gamma induced a rapid tyrosine phosphorylation and nuclear translocation of STAT1 protein, which is involved in the binding to the GAS-2 site in the p11 promoter by EMSA analysis. These data suggest that IFN-gamma-induced p11 expression is mediated through the binding of STAT1 to GAS sites in the p11 promoter. Inhibition of p11 expression by inhibitory antisense RNAs (iRNA) treatment resulted in enhanced IFN-gamma and calcium ionophore-stimulated arachidonic acid release suggesting that at least in part IFN-gamma-stimulated p11 expression may serve a counterregulatory role.

Annexin A2↗
PubMed2003

Divergent nitric oxide bioavailability in men and women with sickle cell disease.

BACKGROUND: Although reduced endothelial nitric oxide (NO) bioavailability has been demonstrated in arteriosclerotic vascular disease, the integrity of this system in sickle cell disease remains uncertain. METHODS AND RESULTS: We measured forearm blood flow in 21 patients with sickle cell disease (hemoglobin SS genotype) and 18 black control subjects before and after intra-arterial infusions of acetylcholine, nitroprusside, and the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Endothelium-dependent vasodilation, measured by the percent increase in flow induced by acetylcholine infusion, was significantly greater than in controls (252+/-37% for patients versus 134+/-24% for controls; P<0.0001). However, there was a large sex difference in blood flow responses between female and male patients (340+/-46% versus 173+/-41%; P=0.035). Similarly, basal NO bioactivity, as measured by the percent decrease in flow induced by L-NMMA, was depressed in male compared with female patients (-17+/-5% versus -34+/-4%; P=0.01), as was the response to nitroprusside (86+/-21% versus 171+/-22%; P=0.008). L-NMMA reduced the blood flow response to acetylcholine in women, but not in men. Sex differences in vascular cell adhesion molecule-1 were appreciated, with significant correlations between levels of soluble vascular cell adhesion molecule-1 and blood flow responses to L-NMMA and nitroprusside (r=0.53, P=0.004 and r=-0.66, P<0.001, respectively). CONCLUSIONS: NO bioavailability and NO responsiveness are greater in women than in men with sickle cell disease and determines adhesion molecule expression. Endothelium-dependent blood flows are largely non-NO mediated in male patients. These results provide a possible mechanism for reported sex differences in sickle cell disease morbidity and mortality and provide a basis for novel pharmacological interventions.

Acetylcholine↗
PubMed2003

An emerging role for nitric oxide in sickle cell disease vascular homeostasis and therapy.

Nitric oxide participates in the compensatory response to chronic vascular injury in patients with sickle cell disease. The authors have found reductions of basal and stimulated nitric oxide production and responses to exogenous nitric oxide in male patients with sickle cell disease. Gender differences in nitric oxide bioavailability are probably caused in part by the protective effects of ovarian estrogen on nitric oxide synthase expression and activity in women. Further, in men, and likely all patients during vaso-occlusive crisis and the acute chest syndrome, nitric oxide is destroyed by increased circulating plasma hemoglobin and superoxide. The combined effects of inhaled nitric oxide gas of improving pulmonary ventilation to perfusion matching and hemodynamics, reducing alveolar and systemic inflammation, and inhibiting circulating plasma hemoglobin (and thus restoring peripheral nitric oxide bioavailability) may modulate the course of the disease, including the frequency and severity of vaso-occlusive crises and acute chest syndrome episodes. Possible effects of chronic nitric oxide-based therapies on erythrocyte density, pulmonary artery pressures, and fetal hemoglobin induction deserve study.

Anemia, Sickle Cell↗
PubMed2003

Hydroxyurea induces fetal hemoglobin by the nitric oxide-dependent activation of soluble guanylyl cyclase.

Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of gamma-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced gamma-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.

Cyclic GMP↗
PubMed2002

Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease.

Although the deleterious vasoconstrictive effects of cell-free, hemoglobin-based blood substitutes have been appreciated, the systemic effects of chronic hemolysis on nitric oxide bioavailability have not been considered or quantified. Central to this investigation is the understanding that nitric oxide reacts at least 1,000 times more rapidly with free hemoglobin solutions than with erythrocytes. We hypothesized that decompartmentalization of hemoglobin into plasma would divert nitric oxide from homeostatic vascular function. We demonstrate here that plasma from patients with sickle-cell disease contains cell-free ferrous hemoglobin, which stoichiometrically consumes micromolar quantities of nitric oxide and abrogates forearm blood flow responses to nitric oxide donor infusions. Therapies that inactivate plasma hemoglobin by oxidation or nitric oxide ligation restore nitric oxide bioavailability. Decompartmentalization of hemoglobin and subsequent dioxygenation of nitric oxide may explain the vascular complications shared by acute and chronic hemolytic disorders.

Adult↗
PubMed2002

Effects of S-nitrosation and cross-linking of hemoglobin on hypoxic pulmonary vasoconstriction in isolated rat lungs.

Free hemoglobin (Hb) and red blood cells augment hypoxic pulmonary vasoconstriction (HPV) by scavenging nitric oxide (NO). S-nitrosation of Hb (SNO-Hb) may confer vasodilatory properties by allowing release of NO during deoxygenation and/or by interaction with small-molecular weight thiols. Likewise, cross-linking of free Hb may limit its vasoconstrictive effect by preventing abluminal movement of the molecule. We compared the effects of free SNO-Hb and Hb intramolecularly cross-linked at the beta-cysteine 93 residue [Bis(maleidophenyl)-polyethylene glycol2000HbA (Bis-Mal-PEGHb)] to those of free oxyHb on pulmonary artery pressure (PAP), HPV, and exhaled NO (eNO) in isolated, perfused rat lungs. Ventilation of lungs with anoxic gas for 5 minutes reduced perfusate PO2 to 11+/-1.0 Torr. Addition of SNO-Hb or Bis-Mal-PEGHb (100 micromol/L) to buffer perfusate increased normoxic PAP and augmented HPV in similar magnitude as free oxyHb, but had no effect on eNO. Addition of the allosteric modulator inositol hexaphosphate to increase Hb P50 and the thiol glutathione (GSH) to allow removal of NO from Hb via transnitrosation to the perfusate did not reduce augmentation of HPV by SNO-Hb or increase eNO. GSH resulted in an approximately 50% reduction in perfusate [S-nitrosothiol], in association with an increase in perfusate [metHb]. Free SNO-Hb is a net NO scavenger and pulmonary vasoconstrictor in this model, although thiol-mediated release of NO from SNO-Hb does occur. However, release of NO from SNO-Hb was not influenced by deoxygenation-mediated allosteric changes in Hb across a broad range of oxyHb saturation. Cross-linking of Hb does not limit its pulmonary vasoconstrictor effects.

Animals↗
PubMed2002

Epidermal growth factor induces p11 gene and protein expression and down-regulates calcium ionophore-induced arachidonic acid release in human epithelial cells.

p11, a member of the S-100 family of proteins, is the cellular ligand of annexin II and also interacts with the C-terminal region of cytosolic phospholipase A(2) (cPLA(2)), inhibiting cPLA(2) activity and arachidonic acid (AA) release. It has been reported that epidermal growth factor (EGF) induces cPLA(2) activation or cPLA(2) expression and subsequent AA release. It was of interest to study the effect of EGF on p11 production and on AA release in human epithelial cells (HeLa). EGF (20 ng/ml) treatment of HeLa cells increased the cellular p11 protein and the steady-state levels of p11 mRNA in a time- and dose-dependent manner but did not affect cPLA(2) protein expression over a 4-48-h incubation time. Transient transfection experiments of a reporter gene construct containing 1498 bp of the 5'-flanking region of p11 promoter demonstrated that EGF induced p11 gene expression at the transcriptional level. EGF caused a rapid phosphorylation of p44/42 and p38 kinases with a maximum level at 10 min. AG 1478 (EGF receptor tyrosine kinase inhibitor), PD 98059 (ERK1/2 inhibitor), and SB 203580 (p38 inhibitor) significantly inhibited EGF-induced p11 expression. EGF-induced AA release was significantly suppressed by AG 1478, PD 98059, SB 203580, and methyl arachidonyl fluorophosphate (a specific cPLA(2) inhibitor). Methyl arachidonyl fluorophosphate (50 microm) also significantly inhibited EGF-induced p11 expression, demonstrating that the activation of cPLA(2) may have a role in the EGF-induced p11 expression. Immunoprecipitation experiments showed that EGF induced increased p11 binding to cPLA(2) in a time- and dose-dependent manner. EGF treatment for 30 min increased -induced AA release, whereas EGF treatment for 24 h inhibited -induced AA release. These results suggest that EGF treatment increased p11 bound to cPLA(2) may lead to the late suppression of AA release induced by EGF.

Annexin A2↗
PubMed2002

Effects of iron nitrosylation on sickle cell hemoglobin solubility.

One mechanism by which nitric oxide (NO) has been proposed to benefit patients with sickle cell disease is by reducing intracellular polymerization of sickle hemoglobin (HbS). In this study we have examined the ability of nitric oxide to inhibit polymerization by measuring the solubilizing effect of iron nitrosyl sickle hemoglobin (HbS-NO). Electron paramagnetic resonance spectroscopy was used to confirm that, as found in vivo, the primary type of NO ligation produced in our partially saturated NO samples is pentacoordinate alpha-nitrosyl. Linear dichroism spectroscopy and delay time measurements were used to confirm polymerization. Based on sedimentation studies we found that, although fully ligated (100% tetranitrosyl) HbS is very soluble, the physiologically relevant, partially ligated species do not provide a significant solubilizing effect. The average solubilizing effect of 26% NO saturation was 0.045; much less than the 0.15 calculated for the effect of 26% oxygen saturation. Given the small amounts of NO-ligated hemoglobin achievable through any kind of NO therapy, we conclude that NO therapy does not benefit patients through any direct solubilizing effect.

Electron Spin Resonance Spectroscopy↗
PubMed2002

S-Nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function.

Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an in vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 +/- 17 nm in human arterial erythrocytes and 69 +/- 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the beta-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.

Allosteric Regulation↗
PubMed2002

Haemoglobin: NO transporter, NO inactivator or NOne of the above?

The structural and functional characterization of haemoglobin (Hb) exceeds that of any other mammalian protein. Recently, the biological role attributed to Hb has been extended from the classical role in the transport and exchange of the respiratory gases O(2) and CO(2) to include a third gaseous molecule, nitric oxide (NO). It is postulated that Hb might be involved in the systemic transport and delivery of NO to tissues and in the facilitation of O(2) release. However, definitive evidence for these putative activities is yet to be produced and many questions remain. Here we describe the present status of these hypotheses and their strengths and weaknesses.

Animals↗
PubMed2002

Nitric oxide donor properties of hydroxyurea in patients with sickle cell disease.

Hydroxyurea therapy reduces the rates of vaso-occlusive crisis in patients with sickle cell anaemia and recent data suggest that hydroxyurea treatment can generate nitric oxide (NO). Nitric oxide has been proposed as a novel therapy for sickle cell disease via a number of pathways. We therefore sought to determine whether hydroxyurea has NO donor properties in patients with sickle cell anaemia and explore potential mechanisms by which NO production could be therapeutic. Venous blood was collected from 19 fasting sickle cell anaemia patients, on chronic hydroxyurea therapy, at baseline and 2 and 4 h after a single morning dose of hydroxyurea, as well as 10 patients not taking hydroxyurea. The plasma and red cell NO reaction products nitrate, nitrite and nitrosylated- haemoglobin were measured using ozone-based chemiluminescent assays (using vanadium, KI and I3- reductants respectively). Consistent with NO release from hydroxyurea, baseline levels of total nitrosylated haemoglobin increased from 300 nmol/l to 500 nmol/l (P = 0.01). Plasma nitrate and nitrite levels also significantly increased with peak levels observed at 2 h. Glutathionyl-haemoglobin levels were unchanged, while plasma secretory vascular cellular adhesion molecule-1 levels were reduced in patients taking hydroxyurea (419 +/- 40 ng/ml) compared with control patients with sickle cell anaemia (653 +/- 55 ng/ml; P = 0.003), and were inversely correlated with fetal haemoglobin levels (r = -0.72; P = 0.002). These results demonstrate that hydroxyurea therapy is associated with the intravascular and intraerythrocytic generation of NO. The role of NO in the induction of fetal haemoglobin and possible synergy between NO donor therapy and classic cytostatic and differentiating medications should be explored.

Adolescent↗
PubMed2002

Oxidant-induced cell death in respiratory epithelial cells is due to DNA damage and loss of ATP.

Oxidative stress is considered to be an important pathogenic event in ischemia-reperfusion injury, leading to apoptosis or necrosis. We show acute cytotoxicity upon exposure to hydrogen peroxide (H(2)O(2)) in BEAS-2B cells and A549 cells. Single-cell gel electrophoresis showed formation of large comet tails from DNA upon oxidant exposure suggestive of DNA damage. The ATP content of the cells decreased upon exposure to H(2)O(2). Preincubation with 3-aminobenzamide (3-ABA), an inhibitor of poly (ADP-ribosyl) polymerase (PARP), prevented the cytotoxicity. The decrease in the ATP content of the cells was also prevented by 3-ABA. Increase in PARP activity was further confirmed by measuring incorporation of [(32)P]-NAD into nuclear proteins in presence of the cell extracts. Markers of apoptosis were not seen in cells treated with H(2)O(2) with or without 3-ABA pretreatment. These studies suggest that DNA damage is one of the primary reasons for oxidant-induced cell death and that PARP plays an important role in cell death due to its consumption of ATP. Further elaboration of this and other pathways that consume ATP may help prevent oxidant-mediated acute lung injury.

Adenosine Triphosphate↗