Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease.

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

Multiple inducers of endothelial NOS (eNOS) dysfunction in sickle cell disease.

A characteristic aspect of the robust, systemic inflammatory state in sickle cell disease is dysfunction of endothelial nitric oxide synthase (eNOS). We identify 10 aberrant endothelial cell inputs, present in the specific sickle context, that are known to have the ability to cause eNOS dysfunction. These are: endothelial arginase depletion, asymmetric dimethylarginine, complement activation, endothelial glycocalyx degradation, free fatty acids, inflammatory mediators, microparticles, oxidized low density lipoproteins, reactive oxygen species, and Toll-like receptor 4 signaling ligands. The effect of true eNOS dysfunction on clinical testing using flow-mediated dilation can be simulated by two known examples of endothelial dysfunction mimicry (hemoglobin consumption of NO; and oxidation of smooth muscle cell soluble guanylate cyclase). This lends ambiguity to interpretation of such clinical testing. The presence of these multiple perturbing factors argues that a therapeutic approach targeting only a single injurious endothelial input (or either example of mimicry) would not be sufficiently efficacious. This would seem to argue for identifying therapeutics that directly protect eNOS function or application of multiple therapeutic approaches.

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Microparticles in sickle cell anaemia: promise and pitfalls.

Blood from patients with sickle cell disease contains microparticles (MP) derived from multiple cell sources, including red cells, platelets, monocytes and endothelial cells. MPs are of great interest because of their disease associations, their status as promising biomarkers, and the intercellular communications they mediate. To illustrate the likelihood of their relevance in sickle cell disease, we discuss the nature of MP, their profiling in sickle disease, some caveats relevant to their detection, their roles in supporting coagulation and the disparate influences they may exert upon the pathobiology of sickle cell disease.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4ß1, or αVß3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease.

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.

A mechanistic role for DNA methylation in endothelial cell (EC)-enriched gene expression: relationship with DNA replication timing.

Proximal promoter DNA methylation has been shown to be important for regulating gene expression. However, its relative contribution to the cell-specific expression of endothelial cell (EC)-enriched genes has not been defined. We used methyl-DNA immunoprecipitation and bisulfite conversion to analyze the DNA methylation profile of EC-enriched genes in ECs vs nonexpressing cell types, both in vitro and in vivo. We show that prototypic EC-enriched genes exhibit functional differential patterns of DNA methylation in proximal promoter regions of most (eg, CD31, von Willebrand factor [vWF], VE-cadherin, and intercellular adhesion molecule-2), but not all (eg, VEGFR-1 and VEGFR-2), EC-enriched genes. Comparable findings were evident in cultured ECs, human blood origin ECs, and murine aortic ECs. Promoter-reporter episomal transfection assays for endothelial nitric oxide synthase, VE-cadherin, and vWF indicated functional promoter activity in cell types where the native gene was not active. Inhibition of DNA methyltransferase activity indicated important functional relevance. Importantly, profiling DNA replication timing patterns indicated that EC-enriched gene promoters with differentially methylated regions replicate early in S-phase in both expressing and nonexpressing cell types. Collectively, these studies highlight the functional importance of promoter DNA methylation in controlling vascular EC gene expression.

Age sensitizes the kidney to heme protein-induced acute kidney injury.

Age increases the risk for ischemic acute kidney injury (AKI). We questioned whether a similar age-dependent injury occurs following exposure to hemoglobin, a known nephrotoxin. Old mice (~16 mo old), but not young mice (~6 mo old), when administered hemoglobin, exhibited marked elevation in blood urea nitrogen (BUN) and serum creatinine, and acute tubular necrosis with prominent tubular cast formation. The aged kidney exhibited induction of heme oxygenase-1 (HO-1) and other genes/proteins that may protect against heme-mediated renal injury, including ferritin, ferroportin, haptoglobin, and hemopexin. Old mice did not evince induction of HO-2 mRNA by hemoglobin, whereas a modest induction of HO-2 mRNA was observed in young mice. To determine the functional significance of HO-2 in heme protein-induced AKI, we administered hemoglobin to relatively young HO-2(+/+) and HO-2(-/-) mice: HO-2(-/-) mice, compared with HO-2(+/+) mice, exhibited greater renal dysfunction and histologic injury when administered hemoglobin. In addition to failing to elicit a protective system such as HO-2 in response to hemoglobin, old mice exhibited an exaggerated maladaptive response typified by markedly greater induction of the nephrotoxic cytokine IL-6 (130-fold increase vs. 10-fold increase in mRNA in young mice). We conclude that aged mice, unlike relatively younger mice, are exquisitely sensitive to the nephrotoxicity of hemoglobin, an effect attended by a failure to induce HO-2 mRNA and a fulminant upregulation of IL-6. Age thus markedly augments the sensitivity of the kidney to heme proteins, and HO-2 confers resistance to such insults.

Mechanisms of enhanced thrombus formation in cerebral microvessels of mice expressing hemoglobin-S.

The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (ß(s) mice). Enhanced microvascular thrombosis in ß(s) mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from ß(s) mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in ß(s) mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in ß(s) mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Pilot study of vascular health in survivors of osteosarcoma.

BACKGROUND: Cardiovascular-related toxicities have been reported among survivors of osteosarcoma. METHODS: Fasting blood samples from 24 osteosarcoma survivors were analyzed for high-sensitivity C-reactive protein (hsCRP), triglycerides, total cholesterol, high-density lipoprotein (HDL), apolipoprotein-ß, lipoprotein (a), fibrinogen, circulating endothelial cells (CECs), and surface expression of vascular cell adhesion molecule-1 (VCAM-1). Values were compared to subjects in the natural history Coronary Artery Risk Development in Young Adults (CARDIA) cohort study except for CECs and VCAM-1 expression, which were compared to controls studied at the University of Minnesota Lillehei clinical trials unit. PROCEDURE: Survivors (54.2% male), median age 18 years (9-32) at diagnosis, 36.5 years (20-56) at evaluation were treated with a variety of chemotherapeutic exposures, all but one were exposed to doxorubicin (median dose 450 mg/m(2) ; range: 90-645 mg/m(2)), 14 (58.3%) received cisplatin, and 3 (12.5%) were exposed to carboplatin. Two survivors (8.3%) received radiation therapy for disease relapse. Compared to CARDIA subjects, mean hsCRP (3.0 mg/L ± 2.0 vs. 1.6 ± 2.3), triglycerides (151 mg/dl ± 81.7 vs. 95.4 ± 101.3), lipoprotein (a) (34.9 mg/dl ± 17.7 vs. 13.8 ± 22.0), and fibrinogen (315.0 mg/dl ± 49.3 vs. 252.4 ± 61.7) were significantly elevated. The number of CECs (0.47 cells/ml ± 2.5 vs. 0.92 ± 2.5) did not differ while surface expression of VCAM-1 (86.4% ± 34.0 vs. 42.1 ± 33.8) was significantly elevated compared to controls. CONCLUSIONS: Among survivors of osteosarcoma, assessed a median of 14 years from diagnosis, there is evidence of vascular inflammation, dyslipidemia, and early atherogenesis.

Mouse models for studying pain in sickle disease: effects of strain, age, and acuteness.

The clinical management of severe pain associated with sickle cell disease (SCD) remains challenging. Development of an optimal therapy would be facilitated by use of murine model(s) with varying degrees of sickling and pain tests that are most sensitive to vaso-occlusion. We found that young (≤3 months old) NY1DD and S+S(Antilles) mice (having modest and moderate sickle phenotype, respectively) exhibited evidence of deep tissue/musculoskeletal pain. Deep tissue pain and cold sensitivity in S+S(Antilles) mice increased significantly with both age and incitement of hypoxia/reoxygenation (H/R). C57/BL6 mice (genetic background strain of NY1DD and S+S(Antilles) ) were hypersensitive to mechanical and heat stimuli, even without the sickle transgene. H/R treatment of HbSS-BERK mice with severe sickle phenotype resulted in significantly decreased withdrawal thresholds and enhanced mechanical, thermal and deep tissue hyperalgesia. Deep hyperalgesia incited by H/R in HbSS-BERK was ameliorated by CP 55940, a cannabinoid receptor agonist. Thus, assessment of deep tissue pain appears to be the most sensitive measure for studying pain mechanisms across mouse models of SCD, and HbSS-BERK mice may be the best model for vaso-occlusive and chronic pain of SCD.

Pulmonary hypertension and nitric oxide depletion in sickle cell disease.

During the past decade a large body of experimental and clinical studies has focused on the hypothesis that nitric oxide (NO) depletion by plasma hemoglobin in the microcirculation plays a central role in the pathogenesis of many manifestations of sickle cell disease (SCD), particularly pulmonary hypertension. We have carefully examined those studies and believe that the conclusions drawn from them are not adequately supported by the data. We agree that NO depletion may well play a role in the pathophysiology of other hemolytic states such as paroxysmal nocturnal hemoglobinuria, in which plasma hemoglobin concentrations are often at least an order of magnitude greater than in SCD. Accordingly, we conclude that clinical trials in SCD designed to increase the bioavailability of NO or association studies in which SCD clinical manifestations are related to plasma hemoglobin via its surrogates should be viewed with caution.

Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids.

Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of mu opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.

The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice.

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Pilot study of vascular health in survivors of Hodgkin lymphoma.

BACKGROUND: Vascular-related toxicities have been reported among survivors of Hodgkin lymphoma (HL), but their genesis is not well understood. PROCEDURE: Fasting blood samples from 25 previously irradiated HL survivors were analyzed for biomarkers that can reveal underlying inflammation and/or endothelial cell activation: high-sensitivity C-reactive protein (hsCRP), triglycerides, total cholesterol, high-density lipoprotein (HDL), apolipoprotein ß, lipoprotein (a), fibrinogen, circulating endothelial cells (CECs), and vascular cell adhesion molecule-1 (VCAM-1) expression. Values were compared to subjects in the Coronary Artery Risk Development in Young Adults (CARDIA) study. CECs and VCAM-1 were compared to healthy controls. RESULTS: Survivors (76% male), median age 17.6 years (5-33) at diagnosis, 33.0 years (19-55) at follow-up, included stages IA (n = 6), IIA (n = 10), IIB (n = 2), IIIA (n = 4), and IVA (n = 3) patients. Twenty-four received at least chest radiation therapy (RT) (median dose 3,150 cGy; range: 175-4,650 cGy), one received neck only; 14 (56%) had a history of anthracycline exposure (median dose: 124 mg/m(2) range: 63-200 mg/m2). Compared to CARDIA subjects, mean hsCRP (3.0 mg/L ± 2.0 vs. 1.6 ± 1.9), total cholesterol (194.1 mg/dl ± 33.2 vs. 179.4 ± 32.9), lipoprotein (a) (34.2 mg/dl ± 17.5 vs. 13.8 ± 17.5), and fibrinogen (342.0 mg/dl ± 49.1 vs. 252.6 ± 48.4) were significantly elevated. CECs (2.3 cells/ml ± 1.5 vs. 0.34 ± 1.4) were significantly elevated compared to controls. No difference in VCAM-1 expression (51.1% ± 36.8 vs. 42.3 ± 35.6) was detected. CONCLUSION: HL survivors exposed to RT have evidence of vascular inflammation, dyslipidemia, and injury suggestive of early atherogenesis.

Relationship of Endothelial Microparticles to Obesity and Cardiovascular Disease Risk in Children and Adolescents.

Background Microparticles and endothelial microparticles (EMPs) are implicated in accelerating cardiovascular disease (CVD); however, data in pediatrics are limited. We examined the relationship of microparticles and EMPs with adiposity and subclinical CVD risk measures in a pediatric population to determine their potential as biomarkers of CVD risk. Methods and Results A cross-sectional study of youth (n=280; ages 8-20 years) with a range of body mass index categories was used. Microparticles, EMPs, and activated EMPs were measured by flow cytometry. %Body fat and %visceral adipose tissue were measured by dual X-ray absorptiometry. Measures of arterial stiffness and vascular wall structure were obtained. Linear regression (with log-transformed outcomes) and logistic regression were used to evaluate associations and all results were exponentiated. Youth with overweight/obesity and severe obesity had 2.50 (95% CI, 1.56-4.01) and 3.42 (95% CI, 2.15-5.43) times the geometric means of the total number of microparticles, respectively, compared with those with normal weight. Youth with overweight/obesity and severe obesity had 1.97 (95% CI, 1.09-3.55) and 2.34 (95% CI, 1.31-4.19) times the geometric means of the total number of EMPs, respectively, compared with those with normal weight. There were positive associations between the levels of both microparticles and EMPs with higher adiposity measures and poor CVD risk measures. Youth with higher adiposity showed 1.84 times the odds of having high levels of activated EMPs (%) (odds ratio, 1.84; 95% CI, 1.08-3.14) compared with those with normal weight. Conclusions Levels of microparticles, EMPs, and activated EMPs were positively associated with adiposity and poor subclinical CVD risk in a pediatric population.