Resolution of Sickle Cell Crisis Following Administration of Amiodarone.

Sickle cell disease is a prevalent hematologic condition, but some of the factors that lead to erythrocyte sickling are not fully known. A 58-year-old male patient with a history of sickle cell disease (SCD) and paroxysmal atrial fibrillation was transferred from an outside hospital for further management of refractory sickle cell crisis with acute chest syndrome. Before transfer, the patient received antibiotics and multiple packed red blood cell (pRBC) transfusions, with minimal effect on symptoms or anemia. After transfer, the patient developed rapid supraventricular tachycardia and atrial fibrillation (rates >160) with a drop in blood pressure. He was started on IV amiodarone. His heart rate was subsequently better controlled and converted to sinus rhythm the following day. Three days following initiation of amiodarone, the patient, with a hemoglobin count of 6.4 g/dl, required one additional unit of pRBC. On the fourth day, the patient's hemoglobin count rose to 9.4 g/dl, and he reported a marked improvement in symptoms. The improvements in symptoms and hemoglobin count were sustained, and the patient was discharged two days later. This remarkable improvement in anemia and symptoms triggered a search for potential causes. Amiodarone is a complex drug shown to have effects on multiple cell types, including erythrocytes. A recent preclinical study demonstrated reduced sickling and improved anemia in a murine model of SCD. This case report raises the possibility that amiodarone may have contributed to the rapid improvement in anemia and should be further explored in clinical trials. LEARNING POINTS: Prior studies support a link between erythrocyte sickling and membrane lipid composition.Amiodarone may impact erythrocyte pathophysiology by increasing cellular lipids including bis(mono)acylglycerol phosphate (BMP).Drugs with effects on erythrocyte lipid fractions may be beneficial during sickle cell crises.

Beta1-receptor blockade attenuates atherosclerosis progression following traumatic brain injury in apolipoprotein E deficient mice.

Traumatic brain injury (TBI) is associated with cardiovascular mortality in humans. Enhanced sympathetic activity following TBI may contribute to accelerated atherosclerosis. The effect of beta1-adrenergic receptor blockade on atherosclerosis progression induced by TBI was studied in apolipoprotein E deficient mice. Mice were treated with metoprolol or vehicle following TBI or sham operation. Mice treated with metoprolol experienced a reduced heart rate with no difference in blood pressure. Six weeks following TBI, mice were sacrificed for analysis of atherosclerosis. Total surface area and lesion thickness, analyzed at the level of the aortic valve, was found to be increased in mice receiving TBI with vehicle treatment but this effect was ameliorated in TBI mice receiving metoprolol. No effect of metoprolol on atherosclerosis was observed in mice receiving only sham operation. In conclusion, accelerated atherosclerosis following TBI is reduced with beta-adrenergic receptor antagonism. Beta blockers may be useful to reduce vascular risk associated with TBI.

Amiodarone improves anemia in a murine model of sickle cell disease and is associated with increased erythrocyte bis(monoacylglycerol) phosphate.

Sickle cell disease (SCD) is associated with altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 µM) or medroxyprogesterone (6.75 µM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP.

Interleukin-1 receptor antagonism leads to improved anaemia in a murine model of sickle cell disease and is associated with reduced ex vivo platelet-mediated erythrocyte sickling.

Sickle cell disease (SCD) is associated with haemolytic anaemia and secondary activation of leucocytes and platelets, which in turn may further exacerbate haemolysis. As cytokine signalling pathways may participate in this cycle, the present study investigated whether pharmacological blockade of the interleukin-1 receptor (IL-1R) would mitigate anaemia in a murine model of SCD. Within 2 weeks of treatment, reduced markers of haemolysis were observed in anakinra-treated mice compared to vehicle-treated mice. After 4 weeks of anakinra treatment, mice showed increased numbers of erythrocytes, haemoglobin, and haematocrit, along with reduced reticulocytes. Blood from anakinra-treated mice was less susceptible to ex vivo erythrocyte sickling and was resistant to exogenous IL-1ß-mediated sickling. Supernatant generated from IL-1ß-treated platelets was sufficient to promote erythrocyte sickling, an effect not observed with platelet supernatant generated from IL-1R-/- mice. The sickling effect of IL-1ß-treated platelet supernatant was inhibited by a transforming growth factor-ß (TGF-ß) neutralising antibody, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition, and superoxide scavengers, but replicated by recombinant TGF-ß. In conclusion, pharmacological IL-1R antagonism leads to improved anaemia in a murine SCD model. IL-1ß stimulation of platelets promotes erythrocyte sickling. This effect may be mediated by platelet-derived TGF-ß-induced reactive oxygen species generation though erythrocyte NADPH oxidase.

Diversity, Equity, and Inclusiveness in Medicine and Cardiology: Next Steps for JAHA.

We, the Editors of the Journal of the American Heart Association, sincerely regret the publication of the article "Diversity, Inclusion, and Equity: Evolution of Race and Ethnicity Considerations for the Cardiology Workforce in the United States of America From 1969 to 2019".1 We are aware that the publication of this flawed and biased article has caused a great deal of unnecessary pain and anguish to a number of parties, and reflects extremely poorly on us. We fully support the retraction of this article.

Clopidogrel Resistance in a Murine Model of Diet-Induced Obesity Is Mediated by the Interleukin-1 Receptor and Overcome With DT-678.

OBJECTIVE: Clopidogrel is a commonly used P2Y12 inhibitor to treat and prevent arterial thrombotic events. Clopidogrel is a prodrug that requires bioactivation by CYP (cytochrome P450) enzymes to exert antiplatelet activity. Diabetes mellitus is associated with an increased risk of ischemic events, and impaired ability to generate the active metabolite (AM) from clopidogrel. The objective of this study is to identify the mechanism of clopidogrel resistance in a murine model of diet-induced obesity (DIO). Approach and Results: C57BL/6J mice and IL-1R-/- mice were given high-fat diet for 10 weeks to generate a murine model of diet-induced obesity. Platelet aggregation and carotid arterial thrombosis were assessed in response to clopidogrel treatment. Wild-type DIO mice exhibited resistance to antiplatelet and antithrombotic effects of clopidogrel that was associated with reduced hepatic expression of CYP genes and reduced generation of the AM. IL (Interleukin)-1 receptor-deficient DIO (IL1R-/- DIO) mice showed no resistance to clopidogrel. Lack of resistance was accompanied by increased exposure of the clopidogrel AM. This resistance was also absent when wild-type DIO mice were treated with the conjugate of the clopidogrel AM, DT-678. CONCLUSIONS: These findings indicate that antiplatelet effects of clopidogrel may be impaired in the setting of diabetes mellitus due to reduced prodrug bioactivation related to IL-1 receptor signaling. Therapeutic targeting of P2Y12 in patients with diabetes mellitus using the conjugate of clopidogrel AM may lead to improved outcomes.

Increased mitochondrial NADPH oxidase 4 (NOX4) expression in aging is a causative factor in aortic stiffening.

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction.

Traumatic Brain Injury Leads to Accelerated Atherosclerosis in Apolipoprotein E Deficient Mice.

Traumatic brain injury (TBI) has been associated with atherosclerosis and cardiovascular mortality in humans. However the causal relationship between TBI and vascular disease is unclear. This study investigated the direct role of TBI on vascular disease using a murine model of atherosclerosis. Apolipoprotein E deficient mice were placed on a western diet beginning at 10 weeks of age. Induction of TBI or a sham operation was performed at 14 weeks of age and mice were sacrificed 6 weeks later at 20 weeks of age. MRI revealed evidence of uniform brain injury in all mice subjected to TBI. There were no differences in total cholesterol levels or blood pressure between the groups. Complete blood counts and flow cytometry analysis performed on peripheral blood 6 weeks following TBI revealed a higher percentage of Ly6C-high monocytes in mice subjected to TBI compared to sham-treated mice. Mice with TBI also showed elevated levels of plasma soluble E-selectin and bone marrow tyrosine hydroxylase. Analysis of atherosclerosis at the time of sacrifice revealed increased atherosclerosis with increased Ly6C/G immunostaining in TBI mice compared to sham-treated mice. In conclusion, progression of atherosclerosis is accelerated following TBI. Targeting inflammatory pathways in patients with TBI may reduce subsequent vascular complications.

Obesity-induced Endothelial Dysfunction is Prevented by Neutrophil Extracellular Trap Inhibition.

Endothelial dysfunction precedes atherosclerosis and may constitute a critical link between obesity-related inflammation and cardiovascular disease. Neutrophil extracellular traps (NETs) have been shown to promote vascular damage in murine models of autoimmune disease and atherosclerosis. The impact of NETs towards endothelial dysfunction associated with obesity is unknown. Using a diet-induced obesity (DIO) mouse model, this study investigated whether the inhibition or degradation of NETs could reduce the endothelial dysfunction observed in DIO mice. Following induction of DIO, there were elevated plasma concentrations of monocyte chemoattractant protein-1 (MCP-1) and impairment of mesenteric arteriolar vasorelaxation in response to acetylcholine as measured by pressure myography. A marker of NET formation, cathelicidin-related antimicrobial peptide (CRAMP), was markedly increased in mesenteric arterial walls of DIO mice compared to mice on standard chow. Prevention of NET formation with Cl-amidine or dissolution of NETs with DNase restored endothelium-dependent vasodilation to the mesenteric arteries of DIO mice. These findings suggest an instrumental role for NETs in obesity-induced endothelial dysfunction.

Angiotensin II-induced Hypertension is Reduced by Deficiency of P-selectin Glycoprotein Ligand-1.

Identification of inflammatory mediators that regulate the vascular response to vasopressor molecules may aid in the development of novel therapeutic agents to treat or prevent hypertensive vascular diseases. Leukocytes have recently been shown to be capable of modifying blood pressure responses to vasopressor molecules. The purpose of this study was to test the hypothesis that deficiency of the leukocyte ligand, Psgl-1, would reduce the pressor response to angiotensin II (Ang II). Mice deficient in Psgl-1 (Psgl-1-/-) along with wild-type (WT) controls were treated for 2 weeks with a continuous infusion of Ang II. No differences in blood pressure between the groups were noted at baseline, however after 5 days of Ang II infusion, systolic blood pressures were higher in WT compared to Psgl-1-/- mice. The pressor response to acute administration of high dose Ang II was also attenuated in Psgl-1-/- compared to WT mice. Chimeric mice with hematopoietic deficiency of Psgl-1 similarly showed a reduced pressor response to Ang II. This effect was associated with reduced plasma interleukin-17 (IL-17) levels in Psgl-1-/- mice and the reduced pressor response was restored by administration of recombinant IL-17. In conclusion, hematopoietic deficiency of Psgl-1 attenuates Ang II-induced hypertension, an effect that may be mediated by reduced IL-17.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) Deficiency is Protective Against Venous Thrombosis in Mice.

The effect of lipid lowering on the incidence of deep venous thrombosis (DVT) is controversial. The purpose of this study was to determine the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) deficiency on development of DVT in mice. Pcsk9 deficient (pcsk9 -/-) and wild-type (WT) littermates underwent partial inferior vena cava (IVC) ligation to induce venous thrombosis. 48 hours following IVC ligation, IVC thrombosis was evident in 60% of WT mice and 25% of pcsk9 -/- mice (p < 0.05). Analysis of IVC thrombosis revealed greater thrombus weight, length, myeloid cell recruitment, and more neutrophil extracellular trap formation (NETs) in WT compared to pcsk9 -/- mice. Intravital microscopy performed two hours following partial IVC ligation revealed that leukocyte firm attachment was increased in WT mice compared to mice undergoing a sham operation, however leukocyte attachment was reduced in pcsk9 -/- mice compared to WT mice. In conclusion, deficiency of PCSK9 is associated with protection from venous thrombosis. This protection is associated with reduced leukocyte recruitment and NET formation at the site of thrombosis.

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target.

Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils - neutrophil extracellular traps (NETs), in particular - in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1-KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1-deficient mice by infusion of WT neutrophils, while an anti-PSGL-1 monoclonal antibody inhibited APS IgG-mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS.

Membrane-Tethered Metalloproteinase Expressed by Vascular Smooth Muscle Cells Limits the Progression of Proliferative Atherosclerotic Lesions.

BACKGROUND: The MMP (matrix metalloproteinase) family plays diverse and critical roles in directing vascular wall remodeling in atherosclerosis. Unlike secreted-type MMPs, a member of the membrane-type MMP family, MT1-MMP (membrane-type 1 MMP; MMP14), mediates pericellular extracellular matrix degradation that is indispensable for maintaining physiological extracellular matrix homeostasis. However, given the premature mortality exhibited by MT1-MMP-null mice, the potential role of the proteinase in atherogenesis remains elusive. We sought to determine the effects of both MT1-MMP heterozygosity and tissue-specific gene targeting on atherogenesis in APOE (apolipoprotein E)-null mice. METHODS AND RESULTS: MT1-MMP heterozygosity in the APOE-null background (Mmp14+/-Apoe-/- ) significantly promoted atherogenesis relative to Mmp14+/+Apoe-/- mice. Furthermore, the tissue-specific deletion of MT1-MMP from vascular smooth muscle cells (VSMCs) in SM22α-Cre(+)Mmp14F/FApoe-/- (VSMC-knockout) mice likewise increased the severity of atherosclerotic lesions. Although VSMC-knockout mice also developed progressive atherosclerotic aneurysms in their iliac arteries, macrophage- and adipose-specific MT1-MMP-knockout mice did not display this sensitized phenotype. In VSMC-knockout mice, atherosclerotic lesions were populated by hyperproliferating VSMCs (smooth muscle actin- and Ki67-double-positive cells) that were characterized by a proinflammatory gene expression profile. Finally, MT1-MMP-null VSMCs cultured in a 3-dimensional spheroid model system designed to mimic in vivo-like cell-cell and cell-extracellular matrix interactions, likewise displayed markedly increased proliferative potential. CONCLUSIONS: MT1-MMP expressed by VSMCs plays a key role in limiting the progression of atherosclerosis in APOE-null mice by regulating proliferative responses and inhibiting the deterioration of VSMC function in atherogenic vascular walls.

Hematopoietic Deficiency of miR-223 Attenuates Thrombosis in Response to Photochemical Injury in Mice.

Some studies have shown that levels of MicroRNA (miR)-223 derived from platelets in the plasma are reduced following inhibition of platelet function, while others have shown a correlation between low plasma miR-223 and high on-treatment platelet reactivity. The present study seeks to investigate the role of miR-223 in arterial thrombosis. A model of photochemical-induced carotid thrombosis was applied to miR-223 deficient mice and littermate (WT) controls. Mice deficient in miR-223 exhibited significantly prolonged times to occlusive thrombosis compared to WT mice indicating a protective effect of miR-223 deficiency. Bone marrow transplantation experiments confirmed that the hematopoietic pool of miR-223 was responsible for differences in thrombosis times. Transfusion of either WT platelets or extracellular vesicles derived from WT platelets were both sufficient to shorten thrombosis times in miR-223 deficient recipients. The effect of platelet transfusions on IGF-1R was explored. These experiments revealed that vascular IGF-1R was down-regulated by platelet miR-223. Furthermore, inhibition of IGF-1R abolished the protection conferred by miR-223 deficiency on thrombosis. In conclusion, platelet miR-223 is a regulator of arterial thrombosis following endothelial injury through effects on vascular wall IGF-1R. This study indicates that platelet miR-223 is a potential therapeutic target for prevention of arterial thrombosis.

T-Cells Specific for a Self-Peptide of ApoB-100 Exacerbate Aortic Atheroma in Murine Atherosclerosis.

On the basis of mouse I-Ab-binding motifs, two sequences of the murine apolipoprotein B-100 (mApoB-100), mApoB-1003501-3515 (designated P3) and mApoB-100978-992 (designated P6), were found to be immunogenic. In this report, we show that P6 is also atherogenic. Immunization of Apoe-/- mice fed a high-fat diet (HFD) with P6 resulted in enhanced development of aortic atheroma as compared to control mice immunized with an irrelevant peptide MOG35-55 or with complete Freund's adjuvant alone. Adoptive transfer of lymph node cells from P6-immunized donor mice to recipients fed an HFD caused exacerbated aortic atheromas, correlating P6-primed cells with disease development. Finally, P6-specific T cell clones were generated and adoptive transfer of T cell clones into recipients fed an HFD led to significant increase in aortic plaque coverage when compared to control animals receiving a MOG35-55-specific T cell line. Recipient mice not fed an HFD, however, did not exhibit such enhancement, indicating that an inflammatory environment facilitated the atherogenic activity of P6-specific T cells. That P6 is identical to or cross-reacts with a naturally processed peptide of ApoB-100 is evidenced by the ability of P6 to stimulate the proliferation of T cells in the lymph node of mice primed by full-length human ApoB-100. By identifying an atherogenic T cell epitope of ApoB-100 and establishing specific T cell clones, our studies open up new and hitherto unavailable avenues to study the nature of atherogenic T cells and their functions in the atherosclerotic disease process.

Significant Improvement of Antithrombotic Responses to Clopidogrel by Use of a Novel Conjugate as Revealed in an Arterial Model of Thrombosis.

Clopidogrel is a prodrug that requires bioactivation by cytochrome P450 (P450) enzymes to a pharmacologically active metabolite for antiplatelet action. The clinical limitations of clopidogrel are in large part due to its poor pharmacokinetics resulting from inefficient bioactivation by P450s. In this study, we determined the pharmacokinetics and pharmacodynamics of a novel conjugate of clopidogrel, referred to as ClopNPT, in animal models and we evaluated its potential to overcome the limitations of clopidogrel. Results from pharmacokinetic (PK) studies showed that ClopNPT released the active metabolite with a time to maximal plasma concentration of <5 minutes in C57BL/6 mice after either oral or intravenous administration, and plasma concentrations of the active metabolite reached Cmax values of 1242 and 1100 ng/ml after a 10-mg/kg oral dose and a 5-mg/kg intravenous dose, respectively. Furthermore, ClopNPT was highly effective in preventing arterial thrombosis in rabbits and mice after vascular injuries. Formation of occlusive thrombi was prevented by ClopNPT at the 1-mg/kg dose with no significant increase in tongue bleeding time, whereas clopidogrel was ineffective at the same dose. These results suggest that ClopNPT has favorable PK/pharmacodynamic properties that can potentially overcome the attenuated PK properties of clopidogrel and thus significantly improve the efficacy of antiplatelet therapy.

Psgl-1 Deficiency is Protective against Stroke in a Murine Model of Lupus.

Systemic lupus erythematosus (SLE) is associated with an elevated risk of vascular complications, including premature stroke. Therapies targeting leukocyte recruitment may be beneficial in reducing vascular complications associated with SLE. Lupus was induced in female wild-type (WT) and P-selectin glycoprotein ligand-1 deficient (Psgl-1(-/-)) mice with pristane. Stroke was induced following photochemical injury to the middle cerebral artery (MCA). Stroke size was increased in pristane-treated WT mice compared to non-pristane-treated WT controls. However, stroke size was not increased in pristane-treated Psgl-1(-/-) mice compared to controls, despite evidence of increased nephritis in Psgl-1(-/-) mice. Pristane-treated WT mice showed elevated anti-dsDNA, anti-snRNP, CXCL1, and MCP-1 levels compared to untreated mice; however levels of anti-snRNP, MCP-1, and CXCL1 were reduced in pristane-treated Psgl-1(-/-) mice compared to pristane-treated WT mice. Infiltration of neutrophils and macrophages at the cerebral infarction site were reduced in pristane-treated Psgl-1(-/-) mice compared to pristane-treated WT mice. In conclusion, the increase in stroke size associated with lupus is prevented by Psgl-1 deficiency while nephritis is exacerbated. Therapies targeting Psgl-1 may be useful in the management of SLE patients at high risk of acute vascular complications although elucidation of downstream pathways will be necessary to identify targets that do not promote nephritis.

mTOR Inhibition improves anaemia and reduces organ damage in a murine model of sickle cell disease.

Mechanistic target of rapamycin (mTOR) has been shown to play an important role in red blood cell physiology, with inhibition of mTOR signalling leading to alterations in erythropoiesis. To determine if mTOR inhibition would improve anaemia in sickle cell disease (SCD), mice with SCD were treated with the dual mTORC1/2 inhibitor, INK128. One week after daily oral drug treatment, erythrocyte count, haemoglobin, and haematocrit were all significantly increased while reticulocyte counts were reduced. These parameters remained stable during 3 weeks of treatment. Similar effects were observed following oral treatment with the mTORC1 inhibitor, sirolimus. Sirolimus treatment prolonged the lifespan of sickle cell erythrocytes in circulation, reduced spleen size, and reduced renal and hepatic iron accumulation in SCD mice. Following middle cerebral artery occlusion, stroke size was reduced in SCD mice treated with sirolimus. In conclusion, mTOR inhibition is protective against anaemia and organ damage in a murine model of SCD.