• Reduced prothrombin improves survival and ameliorates inflammation and end-organ damage without spontaneous bleeding in sickle cell mice.• An individual procoagulant, prothrombin, represents a novel therapeutic target that can improve sickle cell disease outcome.Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide "gapmer" to Berkeley SCD mice to selectively reduce circulating FII levels to ∼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FII lox/2 mice (constitutively carrying ∼10% normal FII) and FII WT mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies. (Blood. 2015;126(15):1844-1855

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

P lacenta growth factor (PlGF), an angiogenic growth factor of the vascular endothelial growth factor (VEGF) family, has pleiotropic and redundant roles in development but features prominently in various pathological conditions such as ischemia, angiogenesis, inflammation, atherosclerosis, preeclampsia, and diabetic wound healing (1-9). PlGF expression is induced by hypoxia, erythropoietin, and iron; PlGF effects are manifested via binding to its sole cognate receptor, VEGF receptor 1 (VEGFR-1) (10-13).Recent studies have shown that plasma levels of PlGF are abnormally high in patients with hemolytic anemias, such as in sickle cell disease (SCD) (12,14). Moreover, high plasma PlGF levels correlate with increased incidence of vaso-occlusive events in SCD subjects (12). Consistent with these findings, plasma levels of plasminogen activator inhibitor 1 (PAI-1) and endothelin-1 (ET-1) are high in a mouse model of sickle cell disease, namely, Berkeley sickle (BK-SS) mice, which also show high plasma PlGF levels (15, 16). Conversely, PlGF knockout mice exhibit low plasma PlGF levels and low plasma levels of PAI-1 and ET-1 (15, 16). Furthermore, augmentation of erythroid PlGF expression in normal mice to the levels seen in sickle mice results in increased production of endothelin-1 with associated pulmonary changes, as seen in pulmonary hypertension (PH) in SCD (16); these results were corroborated in a study of 123 patients with SCD (16). Thus, studies in vitro and in vivo support the crucial role of PlGF in upregulating the expression of ET-1 in endothelial cells and associated pulmonary changes characteristic of pulmonary hypertension in SCD.We show that PlGF activates endothelial cells to upregulate the expression of genes such as the ET-1 and PAI-1 genes via activation of hypoxia-inducible factor 1 (HIF-1␣), independently of hypoxia (15,17). Moreover, we have shown that posttranscriptional regulation of ET-1 and PAI-1 is achieved by microRNA 199a2 (miR-199a2), which targets the 3= untranslated region (UTR) of HIF-1␣ mRNA (18).The DNM3 opposite strand (DNM3os) gene produces a noncoding RNA (ncRNA) that serves as the precursor of miR-199a2 and miR-214 (18). This locus is located within an intron of DNM3 and is transcribed from the opposite strand, hence, its designation. Transcription of DNM3os and of cotranscribed miR-199a2/miR-214 is greatly attenuated by PlGF, thus allowing unhindered expansion of HIF-1␣ activity and expression of genes, i.e., the ET-1 and PAI-1 genes, requiring this transcription factor, (18). However, the molecular mechanism of PlGF-mediated downregulation of miR-199a2/miR-214 and DNM3os ncRNA is not fully understood.In the present study, we show that repression of DNM3os in response to PlGF required the participation of activated transcription factor 3 (ATF3), which binds to ATF3 response elements in the DNM3os promoter. The ATF3 gene, a stress-inducible gene, has been shown to play important roles in several pathological conditions, including host immunity and cancer (19-21). To de- Citation...

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Patients in organ failure of vascular origin have increased circulating hematopoietic stem cells and progenitors (HSC/P). Plasma levels of angiotensin II (Ang-II), are commonly increased in vasculopathies. Hyperangiotensinemia results in activation of a very distinct Ang-II receptor set, Rho-family GTPase members, and actin in bone marrow endothelial cells (BMEC) and HSC/P, which results in decreased membrane integrin activation in both BMEC and HSC/P, and in HSC/P de-adhesion and mobilization. The Ang-II effect can be reversed pharmacologically and genetically by inhibiting Ang-II production or signaling through BMEC AT2R, HSCP AT1R/AT2R or HSC/P RhoA, but not by interfering with other vascular tone mediators. Hyperangiotensinemia and high counts of circulating HSC/P seen in sickle cell disease (SCD) as a result of vascular damage, is significantly decreased by Ang-II inhibitors. Our data define for the first time the role of Ang-II HSC/P traffic regulation and redefine the hematopoietic consequences of anti-angiotensin therapy in SCD.