BACKGROUND:Despite a shortage of donors for heart transplant (HT) in the United States, most potential donor hearts are discarded. We evaluated the reasons why and their temporal and geographic variation using a US-wide survey of HT clinicians. METHODS:The Donor Heart Study enrolled 4,333 adult potential heart donors in United States from 2015-2020. Separately by donor, each HT center that refused an offer for that donor was surveyed on their reason(s) for refusal. We measured the prevalence of 28 distinct donor-unrelated reasons for refusal and characterized their temporal and geographic variation. RESULTS:Of 14,132 surveys collected-each representing a single declined donor offer-donor-specific, recipient, and mismatch issues were cited in 49.3%, 24.3%, and 38.0%, respectively. Among surveys citing only donor-specific issues, the most common were age, coronary artery disease, left ventricular (LV) systolic dysfunction, and LV hypertrophy; other noncardiac issue(s) (e.g., drug use) were the sole reason in 20.6% of surveys citing only donor-specific issues. Donor age as a reason for offer refusal became increasingly prevalent over time, despite no accompanying change in the prevalence of older (50+ year-old) donors. Reasons for offer refusal varied widely by region, in a manner unexplained by regional differences in objective donor characteristics. CONCLUSIONS:Our findings highlight the subjective nature of donor heart assessment. Further scrutiny of non-evidence based reasons for refusal could reduce discard of viable donor hearts.

BACKGROUND:Most donor hearts offered for heart transplant (HT) in the United States (US) are turned down. We aimed to understand the reasons for this - focusing on those related to the potential recipient and HT center (i.e., donor-unrelated reasons for refusal). METHODS:The Donor Heart Study (DHS) enrolled 4,333 adult potential heart donors in US from 2015 to 2020. Separately by donor, each HT center who refused an offer for that donor was surveyed on their reason(s) for refusal. We measured the prevalences of 18 distinct donor-unrelated reasons for refusal and their association with the timing of offers (weekend vs. weekday). RESULTS:Our analytic sample included 14,132 unique surveys, each representing a declined offer for one of 3,083 donors (mean per donor: 2.56; range: 1-17). Donor-unrelated reasons were cited in 24.3% (n = 3,441) of surveys; among these, recipient issues (i.e., "recipient ill) were most common (cited in 7%) while resource-related issues (e.g., "logistics", "surgeon unavailable") were rare (<1%). Neither showed a significant time trend; however, other reasons ("already considering another offer", "distance too far") did so, with an abrupt uptick after 2018. We found that several donor-related (but no donor-unrelated) reasons for refusal (e.g., left ventricular hypertrophy, social risk behaviors) were significantly more common on weekends. Their "weekend-predominance" was not explained by differences in objective donor characteristics. CONCLUSIONS:Nearly one quarter of donor heart offer refusals are due to donor-unrelated reasons. Weekend-predominant reasons for offer refusal signal the highly subjective nature of donor assessment and warrant further scrutiny.

Background. C1 esterase inhibitor (C1-INH) is a serine protease inhibitor (serpin) in plasma that regulates the complement and contact systems. C1-INH regulates the complement system, primarily by inhibiting the classical and lectin pathways. It acts by inactivating serine proteases C1r and C1s in the classical pathway and mannan-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in the lectin pathway. In addition, C1-INH inhibits activated factor XII (FXIIa), factor XI (FXIa), and kallikrein thus preventing excessive activation of coagulation via the contact pathway. Similarly, antithrombin III (ATIII), another member of the serpin family, inhibits the activity of thrombin and activated factor X (FXa) in the common pathway. ATIII inhibits thrombin-mediated activation of endothelium and the adhesion of sickle red blood cells and leukocytes to endothelium. Complement activation and thrombin have been shown in murine sickle cell disease (SCD) models to promote microvascular stasis and vascular inflammation. Because of these activities, we hypothesized that infusion of C1-INH or ATIII would prevent vaso-occlusive crises (VOC) induced by hypoxia-reoxygenation (H/R) in the Townes SCD mouse model. Methods. Townes SCD (HbSS) mice and normal HbAA control mice were implanted with dorsal skinfold chambers and challenged with H/R to induce VOC. The mice were infused with saline vehicle, purified human C1-INH or ATIII 1 h prior to H/R or 40 minutes after H/R, simulating prevention and treatment of VOC, respectively. Microvascular stasis in subcutaneous venules was measured 0.5, 1, 2, 3, and 4 h after H/R. After the final stasis measurement, a terminal blood sample was collected from the heart into 0.5 M EDTA and platelet-free plasma was isolated and snap-frozen. The lungs and kidneys were excised and placed in optimal cutting temperature medium. The lungs and kidneys were cryosectioned and examined for P-selectin and von Willebrand factor (vWF) expression on blood vessels (CD31+) in the lungs and complement activation deposits (C3 fragments and C5b9) on blood vessels in the kidneys by confocal microscopy after immunofluorescence staining.Results. Microvascular stasis was significantly decreased (P<0.0001) 1, 2, 3 and 4 h after H/R by pretreatment of HbSS mice with C1-INH or ATIII as compared to vehicle. Similarly, when C1-INH or ATIII was infused 40 minutes after H/R, stasis seen at 30 minutes post-H/R was significantly decreased (P<0.0001) at 1, 2, 3, and 4 h post-H/R as compared to vehicle. Adding enoxaparin to ATIII enhanced ATIII protection. Combining C1-INH and ATIII provided maximal benefit. These observations were mechanistically buttressed in the prevention and treatment studies by decreased expression of P-selectin (P<0.001) and vWF (P<0.005) on the pulmonary endothelium and by reduced deposits of C3 fragments (P<0.01) and C5b9 (P<0.05) in the kidneys 4 h after H/R in HbSS mice administered C1-INH or ATT as compared to vehicle. In addition, plasma IL-6 levels 4 h after H/R was decreased (P<0.001) by treatment of VOC with C1-INH or ATIII as compared to vehicle. These data suggest that inhibiting thrombo-inflammatory pathways with serpins may be a viable therapeutic strategy for VOC in SCD.

Introduction Granulocyte-colony stimulating factor (G-CSF) administration is known to induce severe vaso-occlusive crisis (VOC) in patients with sickle cell disease (SCD). G-CSF and neutrophils rise acutely during VOC, whereas an elevated circulating neutrophil count at steady state is a prognostic factor of SCD severity. G-CSF is induced by many inflammatory triggers, including lipopolysaccharides (LPS), tumor necrosis factor alpha (TNFα) and toll-like receptor 4 activation, which provoke VOC in SCD mice. Importantly, G-CSF induces neutrophil extracellular traps (NETs) formation and activates platelets and monocytes, leading to endothelial cell activation and thrombo-inflammation in SCD. We report the first preclinical evidence validating G-CSF receptor (G-CSFR) blockade as a therapeutic target to prevent VOC in SCD. Methods Townes knock-in humanized SCD mice were exposed to hypoxia-reoxygenation (HR) or intravenously (IV) challenged with oxy-hemoglobin (oxy-Hb), LPS, or G-CSF to induce VOC. Some mice were pretreated IV with VR81, an anti-mouse G-CSFR monoclonal antibody (mAb), prior to VOC induction. Blood flow and NETs formation were assessed by intravital microscopy of the skin and lungs. Liver and lung tissues were examined for inflammation by Western blots and immunofluorescence staining, respectively. Gene expression analyses were performed on transcriptomic datasets from the blood of SCD patients with/without VOC and healthy humans with/without treatment with G-CSF and/or CSL324, an anti-human G-CSFR mAb under development. Results A single IV dose of VR81 in mice had a half-life of ~7 days. Full receptor occupancy in neutrophils was immediate, maintained for at least 1 week, and confirmed by the complete inhibition of phosphorylated STAT3, the signal transduction mediator of G-CSFR signaling. G-CSF administered IV to SCD mice dose-dependently induced microvascular vaso-occlusion in the skin. Vaso-occlusion in the skin was prevented by VR81 administration IV 1 hour or 7 days prior to VOC induction with G-CSF, HR, or oxy-Hb. Importantly, VR81 resolved skin vaso-occlusion in SCD mice when administered 40 minutes after VOC induction. Biomarker analysis revealed several effects caused by G-CSFR blockade. In the liver, expression of pro-inflammatory endothelial adhesion molecules VCAM-1, ICAM-1, and E-selectin and nuclear factor-ĸB-phospho-p65 were markedly decreased, while expression of anti-inflammatory heme oxygenase-1 and nuclear localization of nuclear factor erythroid 2-related factor 2 were increased. Intravital microscopy in the lungs revealed that VR81 pretreatment also prevented neutrophil-platelet aggregate-dependent lung vaso-occlusion in oxy-Hb challenged SCD mice, with a reduced number of NETs within the lung and blood circulation. In the lungs, the number of neutrophils, the expression of pro-adhesive Weibel-Palade body P-selectin, and von Willebrand factor were decreased. Long-term administration of VR81 once per week for 5 months reduced signs of chronic organ injury. The weight of the right ventricle was normalized, suggesting a possible reduction in pulmonary hypertension. The white blood cell count was also reduced. Transcriptomic analyses support the translatability of G-CSFR blockade to SCD patients. We previously published a gene signature of differentially expressed genes in blood after G-CSF administration to healthy humans. In SCD patients, this G-CSF gene signature was significantly enriched during VOC vs. baseline (p-value < 10-3) when analyzed using microarray and RNA-Seq datasets. Conclusions Literature and our data strongly support the critical role of G-CSF in thrombo-inflammatory pathobiology in VOC. Because G-CSF activates multiple inflammatory pathways that trigger vaso-occlusion, G-CSFR is a suitable target for drug intervention to prevent VOC. Our data suggest multiple anti-inflammatory effects in SCD, including prevention of NET formation, which provide a plausible mechanism of action for G-CSFR blockade. The known biology of NETs aligns with a role for G-CSFR in SCD. The NETs formation and their rise during VOC have been confirmed from the literature for patients with SCD. CSL324 was safe and well tolerated in repeat-dose toxicological studies in non-human primates and has completed a phase 1 trial, with satisfactory safety (some risk of transient neutropenia) and pharmacokinetic profiles. A phase 2 trial in patients with SCD will seek clinical proof of concept for VOC prevention.

Individuals with JAK2V617F+ myeloproliferative neoplasm (MPN) exhibit symptoms of microvascular stasis that include headaches, itch, erythromelalgia, and visual disturbances. JAK2V617F+ individuals also have increased bleeding and clotting risk. JAK2V617F+ transgenic mice have increased thrombosis; however, bleeding has limited use of traditional thrombosis models. Dorsal skin fold chambers (DSFC) have been used in sickle cell mouse models to assess microvascular stasis. We sought to determine if DSFC could be applied to model microvascular occlusion in JAK2V617F+ mice and to characterize the baseline endothelial-related pro-thrombotic state. Methods: C57BL/6 (control n=10) and JAK2V617F+ transgenic mice which express one (JAK2VF, n=22) or two copies (JAK2VF/VF, n=3) of a human JAK2 (Janus kinase 2) gene carrying the pathological V617F substitution were used.For DFSC implantation, mice were anesthetized, denuded on back, followed by chamber placement and microvessel dissection. Mice were then placed on microscope for vessel enumeration. After recovery, the mice were injected with TNF-α to initiate a pro-inflammatory state. Mice were re-anesthetized and vessel enumeration was completed at 1-4 h post-TNF-α infusion. For n=5 JAK2VF mice, a subcutaneous injection of 5 mg/mL enoxaparin was added 1 h prior to TNF-α infusion. After 4 h timepoint, mice were euthanized. An additional co-hort of mice, including C57BL/6J (n=4), JAK2VF (n=18), and JAK2VF/VF (n=4) were sacrificed for blood counts, plasma cytokine analysis, and pathologic organ assessment. For organ assessment, 6 µm sections of inflated mouse lung were stained for CD142 (tissue factor), von Willebrand factor (VWF) and CD31 with immunofluorescence. Five images per animal (n=4) were taken followed by quantitative pixel count analysis using Python-based Napari image analysis. Results: Ten C57BL6/J mice, 22 JAK2VF, and 3 JAK2VF/VF mice underwent DFSC implantation. There was no difference in procedure-related mortality between mouse genotypes, with 2 deaths in the C57BL6/J (22.2%) and JAK2VF (9.5%) mortality. JAK2VF/VF had no mortality. 1 JAK2VF mouse was excluded from analysis due to incidental pregnancy. For bleeding, no C57BL6/J or JAK2VF/VFmice experienced bleeding; 3 female JAK2VF mice had minor bleeding. A baseline, there was no difference in microvessel occlusion between genotypes. Starting at 1 hour after TNF-α infusion, there was a significant increase in % microvessels occluded in JAK2VF and JAK2VF/VFcompared to C57BL6/J (p<0.05 via Two-Way ANOVA with Tukey's multiple comparisons). Starting at 2 h after TNF-α, compared to respective genotype baseline, JAK2VFand JAK2VF/VFmice had significantly higher % microvessels occluded. In comparison, C57BL6/J did not experience any significant change in microvessel flow at any timepoint. Likewise, enoxaparin treatment of JAK2VF mice prevented microvessel occlusion. Evaluating relationship between microvessel occlusion with age, gender, weight, white blood cell count, hemoglobin, hematocrit, and platelet count variables using least square mean linear regression modeling found that elevated weight was significantly associated increased % occlusion at 2 hours. Thrombin-antithrombin (TAT), a measure of coagulation, was assessed in all mice. Compared to untreated C57BL6/J mice, JAK2VF and JAK2VF/VFmice had significantly higher TAT levels. C57BL6/J and JAK2VF mice treated with TNF-α had significantly increased TAT. Last, quantitative image analysis of mouse lung immunofluorescent staining revealed that compared to C57BL6/J mice, JAK2VF mice had significant increase in endothelial cell-derived von Willebrand factor (VWF) and pro-coagulant tissue factor (TF). Conclusions: Overcoming the limitations of other models, JAK2VFand JAK2VF/VFmice can undergo DSFC implantation without excessive mortality or bleeding. Weight was found to be associated with % microvascular occlusion in JAK2VFmice. At 2 h after TNF-α infusion, JAK2V617F+ mice exhibit increased microvascular occlusion; anticoagulation with enoxaparin prevents occlusion. At baseline, JAK2VF and JAK2VF/VFmice exhibit increased pro-coagulant state that includes elevated TAT complexes and increased lung endothelial staining of VWF and TF. Our data confirm that JAK2VF mice have increased pro-coagulant state and that DFSC can be applied to JAK2V617F+ mice to model microvascular occlusion.

Introduction: Sickle cell disease (SCD) is characterized by episodic vaso-occlusive crises (VOC), chronic hemolysis, and multiorgan dysfunction. VOC has negative effects on quality of life, is a major cause of hospitalization, and has an elevated risk for death. Oxidative stress, inflammation, enhanced adhesion of activated neutrophils, release of neutrophil traps (NET), hypercoagulability, and endothelial activation are increasingly recognized as playing a primary role in vaso-occlusion, tissue ischemia, and organ damage. Activation of TLR4 and NLRP3 inflammasome have been implicated as key inflammatory pathways in SCD pathophysiology. A growing body of evidence suggests multiple roles for Bruton tyrosine kinase (BTK) as a regulator of the innate inflammatory machinery, the NLRP3 inflammasome, heme and TLR4-mediated inflammasome activation, adhesion molecule expression, and NET formation. Our objective was to evaluate the effect of the BTK inhibitor (BTKi) rilzabrutinib on inflammatory and adhesion molecule expression, and prevention of microvascular stasis in a model of vaso-occlusion in SCD mice. Methods: HbSSTownes SCD mice aged 10-14 weeks (n=12/group) were pretreated with the reversible covalent BTKi rilzabrutinib (40 mg/kg twice daily), the irreversible covalent BTKi RA15539667 (15 mg/kg/day once daily), a P-selectin blocking monoclonal antibody (mAb, 200 µg IP weekly), a control mAb (200 µg IP weekly), or vehicle control. Control HbAA mice were pretreated with vehicle. All mice were pretreated for 2 weeks prior to hypoxia/reoxygenation (H/R) or hemoglobin (Hb) challenge.After 13 days of dosing, the final doses of rilzabrutinib (27th dose), RA15539667 (14th dose), or vehicle were administered in the morning, mice were weighed, implanted with a dorsal skinfold chamber (DSFC), and challenged with H/R (7% O2, 93% N2 for 1 hour, followed by return to normoxia) or infusion of human HbA (1 µmol heme/kg body weight) via the tail vein. Flowing venules in the DSFC window were selected at baseline and re-examined for stasis (no flow) at 1, 2, 3, and 4 hours after return to normoxia. The percent stasis was determined for each time point and treatment group. Mice were euthanized in CO2 at 4 hours after the last timepoint prior to tissue and blood collection. Complete blood counts were measured manually in fresh blood. Immunoblots measured liver nuclear NF-ĸB phospho(Ser536) and total p65 expression on nuclear extracts from liver (n=3/treatment/ challenge; total n=36). Lung P-selectin, von Willebrand factor, and endothelial cell marker CD31 expression were examined by immunofluorescence staining and expressed relative to CD31 (n=3/treatment/challenge; total n=36). Transcriptomic analysis was performed using Twist capture exome sequencing on liver extracts of the left lobe. Results: Pretreatment for 14 days with rilzabrutinib or RA15539667 in HbSS mice significantly decreased microvascular stasis at 1, 2, 3, and 4 hours after both H/R or Hb challenge (vs vehicle/control mAb; P<0.01). Rilzabrutinib and RA15539667 significantly decreased spleen weight (P<0.01) and lowered the inflammatory state in HbSS mice as evidenced by the reduction in total white blood cell count (P<0.01). Significant decreases in inflammation were observed in post-challenge tissue samples including decreased activation of NF-κB (P<0.001) in liver and reduced mobilization of P-selectin and von Willebrand factor by immunofluorescence staining of lung tissue after H/R (P<0.05). In liver of SCD mice from the Hb model, treatment with rilzabrutinib resulted in downregulation of inflammasome-related genes (BTK, caspase-1, NLRP3, IL-1b, IL-18). In both H/R and Hb models, rilzabrutinib treatment led to downregulation of gene expression of markers of inflammation, markers of adhesion (P-selectin, E-selectin/C62L), complement, NETosis, and thrombosis. There were no significant differences among treatment groups compared to vehicle for any red blood cell indices. Conclusion: Preclinical data provides evidence that treatment with rilzabrutinib ameliorates inflammation through multiple mechanisms of action and prevents microvascular stasis in Townes SCD mice.

BACKGROUND:Despite a shortage of potential donors for heart transplant in the United States, most potential donor hearts are discarded. We evaluated predictors of donor heart acceptance in the United States and applied machine learning methods to improve prediction. METHODS:We included a nationwide (2005-2020) cohort of potential heart donors in the United States (n=73 948) from the Scientific Registry of Transplant Recipients and a more recent (2015-2020) rigorously phenotyped cohort of potential donors from DHS (Donor Heart Study; n=4130). We identified predictors of acceptance for heart transplant in both cohorts using multivariate logistic regression, incorporating time-interaction terms to characterize their varying effects over time. We fit models predicting acceptance for transplant in a 50% training subset of DHS using logistic regression, least absolute shrinkage and selection operator, and random forest algorithms and compared their performance in the remaining 50% (test) of the subset. RESULTS:Predictors of donor heart acceptance were similar in the nationwide and DHS cohorts. Among these, older age (P value for time interaction, 0.0001) has become increasingly predictive of discard over time while other factors, including those related to drug use, infection, and mild cardiac diagnostic abnormalities, have become less influential (P value for time interaction, <0.05 for all). A random forest model (area under the curve, 0.908; accuracy, 0.831) outperformed other prediction algorithms in the test subset and was used as the basis of a novel web-based prediction tool. CONCLUSIONS:Predictors of donor heart acceptance for transplantation have changed significantly over the last 2 decades, likely reflecting evolving evidence regarding their impact on posttransplant outcomes. Real-time prediction of donor heart acceptance, using our web-based tool, may improve efficiency during donor management and heart allocation.

BACKGROUND:Potential organ donors often exhibit abnormalities on electrocardiograms (ECGs) after brain death, but the physiological and prognostic significance of such abnormalities is unknown. OBJECTIVES:This 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. METHODS:The 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 multivariable regression. RESULTS:Initial ECGs were interpretable for 4,136 potential donors. Overall, 64% of ECGs were deemed clinically abnormal, most commonly as a result of a nonspecific St-T-wave abnormality (39%), T-wave inversion (19%), and/or QTc interval >500 ms (17%). Conduction abnormalities, ectopy, pathologic Q waves, and ST-segment elevations were less common (each present in ≤5% of donors) and resolved on repeat ECGs in most cases. Only pathological Q waves were significant predictors of donor heart nonuse (adjusted OR: 0.39; 95% CI: 0.29-0.53), and none were associated with graft survival at 1 year post-HT. CONCLUSIONS:ECG abnormalities are common in potential heart donors but often resolve on serial testing. Pathologic Q waves are associated with a lower likelihood of use for HT, but they do not portend worse graft survival.

Patients with sickle cell disease (SCD) experience both chronic and acute pain. Acute pain is due to unpredictable episodes of vaso-occlusion, referred to as vaso-occlusive crises (VOCs). Due to its severity, pain from VOC is the primary reason for emergency room visits and hospitalizations. Opioids are the primary treatment for pain from VOC but are associated with many serious side effects. Thus, new and effective analgesics are needed to effectively manage acute VOC pain, prevent hospitalizations, and reduce opioid use in SCD patients. Nitrous oxide (N2O) is an FDA-approved medical gas with well-known analgesic properties. Inhaled N2O is the only delivery platform in clinical use. Unfortunately, there are substantial barriers to using inhaled N2O at home, including: 1) poor dosing control; 2) inadvertent exposure due to leaking from compressed gas cylinders and inhalation equipment; and 3) patient resistance. Oral administration of N2O would circumvent these barriers to home use. HBI-201 is an oral liquid drug product containing N2O that was developed and manufactured by Hillhurst Biopharmaceuticals, Inc. Using humanized transgenic mice with SCD, we determined if oral administration of HBI-201 reduced hyperalgesia produced by VOC. Male and female Townes HbSS (sickle) and HbAA (control) mice that did not exhibit ongoing hyperalgesia were used. VOC was evoked by exposing mice to cold ambient temperature (10oC) for 1 hour. This produces vaso-occlusion that leads to robust mechanical and heat hyperalgesia (Khasabova et al., 2022). Mechanical hyperalgesia was defined as a decrease in paw withdrawal threshold (PWT) using calibrated von Frey monofilaments applied to the plantar hind paw. Heat hyperalgesia was defined as a decrease in paw withdrawal latency (PWL) in response to a radiant heat stimulus applied to the plantar hind paw. PWT and PWL were averaged for both paws. PWT and PWL were determined before and at 10 min after cold exposure. Mice then received vehicle or HBI-201 by gavage (4, 10 or 20 mg/kg body weight, p.o.), or morphine (5 mg/kg, s.c.). PWT and PWL were determined at various times thereafter. Analgesic effects of HBI-201 were also measured in naïve C57 mice. HBI-201, but not vehicle, reduced cold-evoked mechanical and heat hyperalgesia dose-dependently. The anti-hyperalgesic effect of HBI-201 peaked at approximately 90 min after administration and persisted for approximately 3 hours following the highest dose. The anti-hyperalgesia following HBI-201 (20 mg/kg, p.o.) was similar to that produced by morphine (5 mg/kg, s.c.). There were no significant differences between male and female mice. HBI-201 also produced analgesia (increased PWT and PWL) in naive C57 mice that lasted for 1-2 hours. We conclude that oral administration of HBI-201 (N2O) shows promise and potential to reduce pain, hospitalization and opioid utilization in sickle patients experiencing a painful VOC. Reference: Khasabova, I.A., Juliette, J., Rogness, V.M., Khasabov, S.G., Golovko, M.Y., Golovko, S.A., Kiven, S., Gupta, K., Belcher, J.D., Vercellotti, G.M., Seybold, V.S., and Simone, D.A. A model of painful vaso-occlusive crisis in mice with sickle cell disease. Blood, 140(16):1826-1830, 2022.

Sickle cell disease (SCD) is a devastating hemolytic disease, marked by recurring bouts of painful vaso-occlusion, leading to tissue damage from ischemia/reperfusion pathophysiology. Central to this process are oxidative stress, endothelial cell activation, inflammation, and vascular dysfunction. The endothelium exhibits a pro-inflammatory, pro-coagulant, and enhanced permeability phenotype. We used flow cytometry to enumerate circulating endothelial cells (CECs, CD31+/CD45-/CD146+) in SCD and normal healthy control blood samples. Furthermore, we assessed CEC subtypes, including circulating endothelial progenitor cells (EPCs, CD31+/CD45-/CD146+/CD133+) and mature CECs (mCECs, CD31+/CD45-/CD146+/CD133-) with mCECs further subdivided into resting CECs (rCECs, VCAM-1-) and activated CECs (aCECs, VCAM-1+). As compared to healthy controls, total CECs and mCECs were elevated in SCD blood as compared to healthy control blood. Using the same markers along with size-based gating, we also used flow cytometry to enumerate endothelial-derived extracellular vesicles (EEVs) in plasma. We assessed EEV subtypes based on VCAM-1 expression, including activated EEVs (aEEVs, CD31+/CD45-/CD146+/CD133-/VCAM-1+) and resting EEVs (rEEVs, VCAM-1 negative), presumably derived from activated and resting endothelial cells, respectively. aEEVs were elevated in SCD patient plasma as compared to healthy controls. Importantly, in SCD patients, total EEVs and aEEVs were increased during self-reported pain crisis as compared to steady state. Plasma markers of endothelial cell activation including soluble E-selectin, P-selectin, VCAM-1, and ICAM-1 were elevated in SCD plasma. These data highlight strategies to detect SCD-related endothelial cell activation and demonstrate that endothelial cell activation markers may be useful to evaluate curative and non-curative therapies in SCD patients.

Sickle cell disease (SCD) is the most common inherited monogenetic disorder. Chronic and acute pain are hallmark features of SCD involving neural and vascular injury and inflammation. Mast cells reside in the vicinity of nerve fibers and vasculature, but how they influence these structures remains unknown. We therefore examined the mechanism of mast cell activation in a sickle microenvironment replete with cell-free heme and inflammation. Mast cells exposed to this environment showed an explosion of nuclear contents with the release of citrullinated histones, suggestive of mast cell extracellular trap (MCET) release. MCETs interacted directly with the vasculature and nerve fibers, a cause of vascular and neural injury in sickle cell mice. MCET formation was dependent upon peptidylarginine deiminase 4 (PAD4). Inhibition of PAD4 ameliorated vasoocclusion, chronic and acute hyperalgesia, and inflammation in sickle mice. PAD4 activation may also underlie neutrophil trap formation in SCD, thus providing a novel target to treat the sequelae of vascular and neural injury in SCD.

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 TTE. TTEs were also examined for presence of LV regional wall motion abnormalities and their reversibility. We assessed associations between LV dysfunction, donor heart acceptance for transplantation, and recipient 1-year survival. RESULTS: An initial TTE was interpreted for 3794 of the 4333 potential donors by neurologic determination of death. A total of 493 (13%) of these TTEs showed LV dysfunction. Among those donors with an initial TTE, LV dysfunction was associated with younger age, underweight, and higher NT-proBNP (N-terminal pro-B-type natriuretic peptide) and troponin levels. A second TTE was performed within 24±6 hours for a subset of donors (n=224) with initial LV dysfunction; within this subset, 130 (58%) demonstrated reversibility. Sixty percent of donor hearts with normal LV function were accepted for transplant compared with 56% of hearts with reversible LV dysfunction and 24% of hearts with nonreversible LV dysfunction. Donor LV dysfunction, whether reversible or not, was not associated with recipient 1-year survival. CONCLUSIONS: LV dysfunction associated with brain death occurs in many potential heart donors and is sometimes reversible. These findings can inform decisions made during donor evaluation and help guide donor heart acceptance for transplantation.

PDF file - 2219K, CO enhances DNA damage of PCa cells.

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 exposure also produced mechanical hyperalgesia as measured by paw withdrawal threshold in HbSS mice compared with that in HbAA mice or HbSS mice left at room temperature. Vaso-occlusion and hyperalgesia were associated with an increase in complement activation fragments Bb and C5a in plasma of HbSS mice after cold exposure. This was accompanied by an increase in proinflammatory NF-κB activation and VCAM-1 and ICAM-1 expression in the liver. Pretreatment of nonhyperalgesic HbSS mice before cold exposure with anti-C5 or anti-C5aR monoclonal antibodies (mAbs) decreased vaso-occlusion, mechanical hyperalgesia, complement activation, and liver inflammatory markers compared with pretreatment with control mAb. Anti-C5 or -C5aR mAb infusion also abrogated mechanical hyperalgesia in HbSS mice with ongoing hyperalgesia at baseline. These findings suggest that C5a promotes vaso-occlusion, pain, and inflammation during VOE and may play a role in chronic pain.

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 similar in the DHS and nationwide cohorts. A random forest model outperformed other prediction algorithms and the inclusion of previously unmeasured predictors (as captured in the DHS) improved model performance (AUC 0.90). In the nationwide cohort, older donor age has become more predictive of non-acceptance over the last 15 years while other factors - including mild cardiac imaging abnormalities, cocaine use, high troponin, hypertension, and Hepatitis C - have become less influential.ConclusionReal-time prediction of donor heart acceptance may improve efficiency during donor management and allocation. The predictors of donor acceptance for HT have changed significantly over time, highlighting the need to continually re-evaluate and update our model. Despite 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. We 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. Predictors of acceptance for HT were similar in the DHS and nationwide cohorts. A random forest model outperformed other prediction algorithms and the inclusion of previously unmeasured predictors (as captured in the DHS) improved model performance (AUC 0.90). In the nationwide cohort, older donor age has become more predictive of non-acceptance over the last 15 years while other factors - including mild cardiac imaging abnormalities, cocaine use, high troponin, hypertension, and Hepatitis C - have become less influential. Real-time prediction of donor heart acceptance may improve efficiency during donor management and allocation. The predictors of donor acceptance for HT have changed significantly over time, highlighting the need to continually re-evaluate and update our model.

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 increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

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 adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin- or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target.