John D Belcher

BackgroundPotential organ donors often exhibit abnormalities on electrocardiograms (ECGs) after brain death, but the physiological and prognostic significance of such abnormalities is unknown.ObjectivesThis study sought to characterize the prevalence of ECG abnormalities in a nationwide cohort of potential cardiac donors and their associations with cardiac dysfunction, use for heart transplantation (HT), and recipient outcomes.MethodsThe Donor Heart Study enrolled 4,333 potential cardiac organ donors at 8 organ procurement organizations across the United States from 2015 to 2020. A blinded expert reviewer interpreted all ECGs, which were obtained once hemodynamic stability was achieved after brain death and were repeated 24 ± 6 hours later. ECG findings were summarized, and their associations with other cardiac diagnostic findings, use for HT, and graft survival were assessed using univariable and …

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also …

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous …

The present disclosure relates to the use of MASP-2 inhibitors and/or MASP-3 inhibitors and compositions comprising the same for treatment of sickle cell disease, including treatment, reduction, and/or prevention of sickle cell disease symptoms or manifestations.

PurposeDespite a scarcity of potential donors for heart transplantation (HT) in the United States (US), a minority are actually accepted for HT. We evaluated donor characteristics associated with heart acceptance in the US and applied modern analytic methods to improve the prediction of heart acceptance.MethodsWe included potential heart donors in the US from 2005 - 2020 (n = 73,948), a recent subset (n = 4,110, spanning 2015 - 2020) of which was enrolled in the Donor Heart Study (DHS). We identified and compared predictors of acceptance among DHS and other donors using logistic regression, incorporating interaction terms in the non-DHS ("nationwide") cohort to characterize time-varying effects. A prediction model was developed using prospectively-collected donor data in the DHS subset, and implemented in the form of a web-based prediction tool.ResultsPredictors of acceptance for HT were …

Vaso-occlusive pain episodes (VOE) cause severe pain in patients with sickle cell disease (SCD). Vaso-occlusive events promote ischemia/reperfusion pathobiology that activates complement. We hypothesized that complement activation is linked to VOE. We used cold to induce VOE in the Townes sickle homozygous for hemoglobin S (HbSS) mouse model and complement inhibitors to determine whether anaphylatoxin C5a mediates VOE. We used a dorsal skinfold chamber to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paw to assess mechanical hyperalgesia in HbSS and control Townes mice homozygous for hemoglobin A (HbAA) mice after cold exposure at 10°C/50°F for 1 hour. Cold exposure induced more vaso-occlusion in nonhyperalgesic HbSS mice (33%) than in HbAA mice (11%) or HbSS mice left at room temperature (1%). Cold …

BACKGROUND Left ventricular dysfunction in potential donors meeting brain death criteria often results in nonuse of donor hearts for transplantation, yet little is known about its incidence or pathophysiology. Resolving these unknowns was a primary aim of the DHS (Donor Heart Study), a multisite prospective cohort study. METHODS The DHS enrolled potential donors by neurologic determination of death (n=4333) at 8 organ procurement organizations across the United States between February 2015 and May 2020. Data included medications administered, serial diagnostic tests, and transthoracic echocardiograms (TTEs) performed: (1) within 48 hours after brain death was formally diagnosed; and (2) 24±6 hours later if left ventricular (LV) dysfunction was initially present. LV dysfunction was defined as an LV ejection fraction <50% and was considered reversible if LV ejection fraction was >50% on the second …

PurposeElectrocardiographic (ECG) abnormalities are common after brain death in potential organ donors, but their clinical significance is unclear. We assessed the prevalence of ECG abnormalities and their association with cardiac structural and angiographic abnormalities in a large nationwide donor cohort.MethodsWe used data from the Donor Heart Study, which prospectively enrolled brain-dead organ donors across the U.S. from 2015-2020. An ECG and transthoracic echocardiogram (TTE) were performed shortly after brain death for all donors and interpreted by a core reviewer; a coronary angiogram was performed when clinically indicated and interpreted locally. We assessed the prevalence of selected donor ECG abnormalities, their associations with acceptance (vs. non-acceptance) for heart transplant (HT), and whether they are predictive of 1) left ventricular hypertrophy (LVH) on TTE, i.e. ≥ 1.2cm. 2 …

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC‐derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC‐derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV‐mediated short‐term non‐transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC …

PurposeDespite a scarcity of potential donors for heart transplantation (HT) in the United States (US), a minority are actually accepted for HT. We evaluated donor characteristics associated with heart acceptance in the US and applied modern analytic methods to improve the prediction of heart acceptance.MethodsWe included potential heart donors in the US from 2005 - 2020 (n = 73,948), a recent subset (n = 4,110, spanning 2015 - 2020) of which was enrolled in the Donor Heart Study (DHS). We identified and compared predictors of acceptance among DHS and other donors using logistic regression, incorporating interaction terms in the non-DHS ("nationwide") cohort to characterize time-varying effects. A prediction model was developed using prospectively-collected donor data in the DHS subset, and implemented in the form of a web-based prediction tool.ResultsPredictors of acceptance for HT were …

PurposeLeft ventricular (LV) dysfunction after brain death often prompts non-use of potential donor hearts for transplant, but little is known regarding its incidence, reversibility, and impact on outcomes. Shedding light on these unknowns was a primary aim of the Donor Heart Study -a first-of-its-kind, multisite, prospective study of potential heart donors.MethodsWe enrolled potential donors (n = 4333) at 8 organ procurement organizations across the United States from February 2015-May 2020. Data were collected on donor management, serial labs, and diagnostic tests. These included a transthoracic echocardiogram (TTE) performed within 48 hours after brain death and, if LV dysfunction was present, a repeat TTE 24 ± 6 hours later. LV dysfunction was defined as an ejection fraction (LVEF)<50%, as ascertained by a single expert reviewer, and was considered reversible if LVEF was >50% on the second TTE …

Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb …

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80–102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

COI notes: Kalpna Gupta: Honoraria: Tautona Group, Novartis and CSL Behring. Research Grants: Cyclerion, 1910 Genetics, Novartis, Grifols, UCI Foundation, and SCIRE Foundation. None of the other authors have any competing financial interests.

BACKROUND AND AIM: Vaso-occlusive crises (VOC) cause severe pain in patients with sickle cell disease (SCD). To date, epidemiological studies have shown seasonably colder temperatures exacerbate SCD-related pain. Although literature and clinical observations suggest a connection between cold and triggering of acute VOC pain, the biologic mechanisms underlying VOC-evoked pain in SCD patients are poorly understood. Repeated cold or painful events results in vasoconstriction and decreased perfusion, which increases red cell transit time, deoxygenation, and hemoglobin S polymerization [Veluswamy et al. Blood. 2020; 136: 1191]. SCD mice exhibit ischemia-reperfusion (I/R) pathophysiology in response to hypoxia-reoxygenation with a concomitant increase in hyperalgesia, inflammation, and complement activation. Because complement activation plays a crucial role in animal pain models, we used …

Purpose: Endothelial cell (EC) activation and red blood cell (RBC) adhesion are central to both acute and chronic pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles, REVs, are more abundant from SS RBCs compared with HbA-containing RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to transfer heme to endothelial cells and to promote endothelial activation through cell signaling and transcriptional regulation, triggering neutrophil adhesion to activated endothelium in vitro and vaso-occlusion in SS mice in vivo. Our objective is to test the hypothesis that SS REVs will trigger acute non-transcriptional regulation on human microvascular ECs. The adhesion characteristics of SS RBCs to ECs was used as a direct biomarker of endothelial activation.Materials and methods: We examined the impact of SS REVs on microvascular EC activation and RBC …

Introduction: In sickle cell disease (SCD) mouse models, heme activates toll-like receptor 4 (TLR4) MyD88-dependent signaling and activates NOD-like receptor family pyrin domain containing (NLRP) 3 inflammasome assembly, leading to vascular endothelial activation and vaso-occlusion (VO). In SCD patients, cycles of hemolysis-driven VO contribute to acute and chronic pain/hyperalgesia. Several studies have evaluated non-selective histone deacetylase (HDAC) inhibitors for SCD; however, side-effects have limited clinical advancement. HDAC6 is an attractive target as it exhibits cytoplasmic AND nuclear activity and knockout mice lack hematologic consequences. In the cytoplasm, HDAC6 helps assemble NLRP3 inflammasomes and functions downstream of TLR4 to inhibit signaling. In SCD mouse models, TLR4 signaling is also critical for mechanical hyperalgesia. Intriguingly, in non-SCD hyperalgesia …

Solid organ transplantation continues to be constrained by a lack of suitable donor organs. Advances in donor management and evaluation are needed to address this shortage, but the performance of research studies in deceased donors is fraught with challenges. Here we discuss several of the major obstacles we faced in the conduct of the Donor Heart Study—a prospective, multi‐site, observational study of donor management, evaluation, and acceptance for heart transplantation. These included recruitment and engagement of participating organ procurement organizations, ambiguities related to study oversight, obtaining authorization for donor research, logistical challenges encountered during donor management, sustaining study momentum, and challenges related to study data management. By highlighting these obstacles encountered, as well as the solutions implemented, we hope to stimulate further …

Sickle cell disease (SCD) is an inherited recessive hemoglobinopathy that leads to hemolytic anemia, resulting from a point mutation in the beta‐globin chain of hemoglobin (Hb) (glutamic acid 6‐valine) with recurrent painful episodes, significantly shortened life expectancy, and poor quality of life. As compared with the currently licensed therapeutics, the gasotransmitter carbon monoxide (CO) imparts significant protective effects via multiple physiological mechanisms demonstrated in four mouse models of SCD and also in two published clinical studies. The majority of reports of CO administration in both animal and human studies used either iCO or drug products that contain a non‐CO active pharmaceutical ingredient (API) that releases CO, specifically intravenous PEG‐COHb, as the mode of treatment. There has been a great deal of insight into how CO modulates SCD. There are six mechanisms that have been …

Introduction: The sickle mutation of hemoglobin (HbS) afflicts millions of people worldwide and is characterized by hemolytic anemia, inflammation, painful vaso-occlusive crises, significant morbidity, and early mortality. In sickle cell disease (SCD), HbS polymerization under deoxygenation increases red blood cell (RBC) rigidity, adhesivity and susceptibility to lysis. Products of intravascular hemolysis including heme and RBC-released extracellular vesicles (REVs) promote acute and chronic inflammation triggering endothelial activation and abnormal RBC adhesion. Extracellular vesicles (EVs) are nano-or micro-particles composed of a lipid bilayer, comprised of transmembrane proteins, and enclosing intracellular remnants, including cytosolic proteins and miRNAs. EVs are known to exchange biomaterial between cells, and may serve as surrogate markers for the activated state of the parent cell thus are of high …