Transgenic Sickle Mice Research Articles

Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. DisclosuresNo relevant conflicts of interest to declare.

Activation of the Mineralocorticoid Receptor Stimulates Protein Disulfide Isomerase: Role in Sickle Cell Disease

Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. DisclosuresNo relevant conflicts of interest to declare.

Progressive Glomerular Damage in Sickle Cell Trait and Sickle Cell Anemia Mouse Models

▪Homozygous inheritance of the hemoglobin S mutation (Hb SS; sickle cell anemia) affects 1 in 500 African Americans and is consistently associated with an increased risk for kidney disease, although the mechanisms are poorly understood. Heterozygous inheritance (Hb AS; sickle cell trait) affects 1 in 8 African Americans and has also been associated with an increased risk for kidney disease in some, but not all cohorts.We first investigated whether inheritance of the Hb S mutation resulted in incremental kidney damage in Hb AS and Hb SS mice compared to Hb AA mice using transgenic sickle mice >6 months old (Townes model, Jackson Laboratory). Using Masson trichrome stained sections of the kidney, patterns of mesangial expansion were determined in age- and sex-matched mice by a renal pathologist blinded to the hemoglobin genotype of each specimen. Hb AS mice had diffuse (>50% of the glomeruli per slide being involved) mesangial expansion while Hb SS mice had diffuse and global (>50% of the individual glomerulus being involved) mesangial expansion. Glomerular perimeters were measured using NanoZoomer Whole Slide Imaging in 26 randomly selected glomeruli from 2 mice per genotype. Using >300µm in circumference as the definition for an enlarged glomerulus, the proportion of enlarged glomeruli progressively increased from Hb AA to Hb AS to Hb SS mice (Cochran’s test of linear trend, P=0.002)(Figure 1A). Progressively higher urine protein-to-creatinine ratios were also observed from Hb AA to Hb AS to Hb SS mice (Figure 1B; test for linear trend, P=0.03) as were progressively higher kidney weights: Hb AA (429±28mg, n=8), Hb AS (446±27mg, n=18), Hb SS (567±19mg, n=5) (Test for linear trend, P=0.047).We then compared the mRNA expression profiles in the kidney cortices, which predominantly include glomerular and proximal tubular cells, of 15 age- and sex-matched mice (5 per Hb genotype) using the Affymetrix Mouse Gene 2.0ST Array. Using an additive model with an FDR<0.01, 6 of the 10 most differentially expressed genes were involved in heme or iron metabolism (HMOX1: β 0.93, P=0.0004; SLC25A37: β 0.75, P=3.9x10-6), kidney function (SLC4A1: β 0.87, P=1.0x10-5; AQP6: β 0.79, P=2.0x10-5), or inflammation (RSAD2: β 0.76, P=5.2x10-6; C3: β 0.76, P=0.0005). Other genes that have been implicated in kidney disease and were differentially expressed at an FDR<0.01 included PODXL and EFNB2 (encode glomerular proteins integral for maintaining the filtration barrier) as well as FRMD3 and ELMO1 (implicated in diabetic nephropathy). KEGG physiologic pathways among the differentially expressed genes included focal adhesion (3.4-fold, P=6.0x10-7), extracellular matrix-receptor interaction (5.0-fold, P=3.2x10-6), and phosphatidylinositol signaling systems (3.5-fold, P=0.003).In conclusion, we observed progressive glomerular injury, determined by mesangial expansion, proportion of enlarged glomeruli, and urine protein concentrations, in Hb AS and Hb SS mice compared to Hb AA mice. Renal cortex-expressed genes involved in heme and iron homeostasis, kidney function, and inflammation were differentially expressed with inheritance of the Hb S mutation and may play important roles in the pathophysiology of kidney disease in individuals with sickle cell trait or sickle cell disease and will require further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nrf2 Activation Attenuates Hemolysis but Promotes Pulmonary Vascular Dysfunction in Sickle Mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that coordinates the body’s antioxidant and cytoprotective defense against a variety of toxins. Several Nrf2 activators can reactivate gamma globin gene expression and augment fetal hemoglobin production. More recently, genetic and pharmacologic evidence have shown that Nrf2 activation can specifically mitigate the severity of hemolytic anemia, and systemic and local inflammation in transgenic sickle cell disease (SCD) mice. Based on these encouraging results Nrf2 activation has emerged as an attractive therapeutic strategy in SCD. However, the BEACON trial of the Nrf2 activator CDDO-Methyl Ester (CDDO-Me) showed that this therapeutic approach can cause adverse cardiovascular events in patients with chronic kidney disease with comorbid diabetes. Hitherto, the efficacy-toxicity profile generated by individual Nrf2 activating drugs has not been investigated in SCD. There are hundreds of synthetic and naturally occurring Nrf2 activating compounds, and each class of Nrf2 activating compound has a unique pharmacokinetic, pharmacodynamic, toxicokinetic and toxicodynamic profile. We have recently demonstrated that intravascular hemolysis deteriorates with aging in transgenic sickle (SS) mice in a process that can be mitigated by the Nrf2 activator 3H-1,2-dithiole-3-thione (D3T) (Ghosh et al., JCI Insight, 2016). In this study, an in vitro screen of five Nrf2 activating compounds revealed CDDO-Me to be the most potent inducer of cytoprotective enzymes in human pulmonary microvascular endothelial cells. Thus, we performed a long-term prophylactic CDDO-Me treatment of SS mice and examined the effect of the drug on intravascular hemolysis and vascular dysfunction. A cohort of newly weaned SS mice aged ~4 weeks were randomly assigned to receive CDDO-Me (20µmoles/kg/TIW, n=6) or Vehicle (DMSO/TIW, n=10) by oral gavage for 4 months. After the treatment, the total hemoglobin increased by 10% in the CDDO-Me group while it decreased by 5% in the vehicle-treated group (p<0.05). Plasma concentration of VCAM-1 increased in both groups compared to their respective baseline (vehicle p<0.001; CDDO-Me p<0.05). The lack of impact on VCAM-1 by CDDO-Me was contrary to our results with D3T (Ghosh et al., JCI Insight, 2016). To examine this further, we performed vascular reactivity studies on isolated pulmonary arteries. Second order pulmonary arteries were isolated, cut into 2mm rings, and mounted using 40 μm wire. Rings were set at a resting tension equivalent to 100 mmHg transmural pressure. Cumulative dose response curves of endothelin-1 (100 pM-10 nM) and acetylcholine (10nM-100 μM) were used to study vasoconstriction and vasodilation, respectively. Pulmonary arteries isolated from the CDDO-Me treated SS mice showed a 15% reduction in acetylcholine stimulated vasodilation compared to the arteries isolated from the vehicle treated littermates (vehicle n=5, CDDO-Me n=5; p<0.001). In vitro studies of rat smooth muscle cells revealed significant up-regulation of endothelin receptor subtype A (ETA) mRNA by CDDO-Me (p < 0.01, n=6). We obtained similar results of increased ETA expression in rat smooth muscle cells treated with another Nrf2 activator, Dimethyl Fumarate (DMF) (p < 0.01, n=6). Upregulation of endothelin or the ETA is frequently found in pulmonary hypertension. The hypertensive phenotype that we report here in SS mice treated with CDDO-Me is consistent with the adverse cardiovascular events found in the BEACON trial. Together our results show that while CDDO-Me can stabilize and potentially improve hemolytic anemia in SCD, this drug and potentially other Nrf2 activators may promote the development of pulmonary hypertension, which is a major adult complication of SCD. DisclosuresNo relevant conflicts of interest to declare.

Hemoglobin S Oxidation Promotes Plasma-Derived Microparticle Membrane Alterations and Toxicity

Polymerization of sickle cell hemoglobin S (HbS) is recognized as a key event in the pathophysiology of sickle cell disease (SCD). Repeated HbS polymerization promotes an altered red blood cell (RBC) membrane, hemolysis, and microparticle (MP) formation, which have been shown to play significant roles in the interaction of RBCs with vascular endothelium and progression of vaso-occlusive events. Circulating RBC-derived MPs are elevated in SCD patients and they release a significant portion of their contents including oxidized HbS and heme to the cells of the vasculature. We have recently reported that free HbS oxidizes faster, remains locked in a highly oxidizing form (ferryl) longer, and loses heme faster than normal HbA (Kassa et al., J Biol Chem 290: 27939, 2015). The contributions of HbS higher oxidation states (ferric and ferryl heme) to MP formation, membrane alterations, and heme loss are poorly defined in SCD. RBC-derived MPs (ranging in size between 100-300 nm in diameter) generated by sheer stress or isolated by ultracentrifugation from the plasma (circulating) of SCD patients (N=6), ethnically matched control subjects (N=5), humanized transgenic sickle mice (Townes-SS, N=4), and control wild-type mice (Townes-AA, N=4) were identified by flow cytometry using CD235a glycophorin antibody and annexin V for externalized phosphatidylserine (PS). Time courses of Hb oxidation, obtained during 30 hour incubations of mouse or human MPs were biphasic. The initial levels of oxidized (ferric) Hb (30 to 45%) were slightly reduced within the first ~10 hours, likely due to the presence of RBC residual reductive enzymes within MPs. This was followed by a second phase in which Hb oxidation (ferric Hb) increased linearly and uncontrollably to 65 to 75% of total Hb. SCD MP's contained highly reactive ferryl Hb intermediates, carbonylated membrane proteins, and phosphorylated band 3 proteins. Quantitative proteomic analysis indicated a higher level of protein oxidation in MPs derived from SCD mice and patients. Five-fold higher levels of irreversibly oxidized βCys93 oxidation were found in untreated versus hydroxyurea-treated SCD patients. Intriguingly, HbS β subunits from SCD MPs were ubiquitinated and MPs isolated from untreated SCD patients had 25-fold higher ubiquitination levels than hydroxyurea-treated SCD patients that were comparable to normal controls. MP ubiquitination levels were correlated with HbS and an overall increase in MP oxidative stress, and inversely correlated with HbF. Compared to respective control MPs, incubation of either mice or human SCD MPs with human endothelial cells (HUVEC) activated apoptotic pathways and impacted cellular bioenergetic parameters by lowering mitochondrial oxygen consumption rates to a greater degree in a manner that was correlated with the redox state of Hb iron within MPs. Human endothelial cells incubated with SCD MPs showed greater intracellular reactive oxygen species production and heme oxygenase-1 induction. In summary, Hb transformation to higher oxidation forms is markedly increased in MPs generated from SCD mice and patients, which when incubated with endothelial cells, lead to mitochondrial dysfunction and apoptotic cell death. These mechanistic analyses of RBC-derived SCD microparticles suggest potential anti-oxidative reducing modalities that may interrupt MP heme-mediated pathophysiology in patients with SCD. DisclosuresBelcher:Cydan/Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.

A Novel, Highly Potent and Selective PDE9 Inhibitor for the Treatment of Sickle Cell Disease

▪BackgroundHydroxyurea (HU), a chemotherapeutic agent and an approved therapy for sickle cell disease (SCD), increases cGMP levels and thereby increases fetal hemoglobin (HbF) levels in RBC, which generally correlates with decreased hemolysis and reduced disease severity in subjects with SCD. In addition, increased cGMP levels are anti-inflammatory and reduce the adhesion of WBC to the vascular endothelium. However, potential adverse effects of HU such as infertility, susceptibility to infections, or teratogenic effect have been the subject of concerns. Phosphodiesterase-9 inhibitors (PDE9i) may provide an alternative to HU because of their ability to inhibit the degradation of cGMP and increase cellular cGMP levels. Previously described PDE9i were developed to inhibit neuronal PDE9 for neurologic diseases, like Alzheimer's. A PDE9i with low brain penetrance may be preferable for the chronic treatment of SCD adults and children. IMR-687 is a safe, potent, PDE9i being developed specifically for SCD without these liabilities of HU or the high brain penetrance of other PDE9is.HypothesisWe hypothesized that PDE9i's will increase cellular cGMP levels in hematopoietic cells, increase HbF in red blood cells (RBC), inhibit RBC sickling, decrease inflammation (leukocytosis), and inhibit vaso-occlusion in murine models of SCD.Results & MethodsIMR-687, is a potent inhibitor of PDE9A (IC50 <10 nM) and demonstrates >100-fold higher IC50 with other phosphodiesterases. To determine the ability of IMR-687 to induce cGMP, K562 erythroid cells were exposed to increasing concentrations of IMR-687 or HU and cGMP assessed at 6 hours. IMR-687, at much lower drug concentrations, induced higher levels of cGMP than HU at 6 hours (Fig. Left Panel).We next tested IMR-687 in the Berkeley (Berk) mouse model of SCD. Groups of 7-8 Berk SCD mice were dosed by gavage with either vehicle, 30 mg/kg/day of IMR-687, or 100 mg/kg/day of HU. After 30 days of treatment, both IMR-687 and HU resulted in statistically significant decreases in the percentage of sickled RBCs (Fig. Center Left Panel) and increases in the percentage of HbF positive RBCs relative to controls. In addition, both compounds led to statistically significant decreases in total bilirubin, spleen weight and total leucocyte counts (Fig. Center Right Panel).The ability of IMR-687 to reduce microvascular stasis (% non-flowing venules) was assessed in HbSS-Townes transgenic sickle mice after transient hypoxia and re-oxygenation. Groups of 3 mice/group were dosed orally via drinking water with IMR-687 (30 mg/kg/day), HU (100 mg/kg/day) or vehicle for 10 days. After 10 days of treatment, the mice were exposed to transient hypoxia. Compared with controls, 30 mg/kg/day IMR-687 and 100 mg/kg HU produced statistically significant reductions in microvascular stasis at both 1-hour (Fig. Center Right Panel) and 4-hour time points post-hypoxia. In addition, IMR-687 significantly reduced the proportion of sickled RBCs, increased the number of HbF positive red cells, and reduced the white count.Lastly, low brain penetration of IMR-687 was confirmed after IV dosing in the rat, with brain concentrations >20 times lower than those in the plasma at all time points assessed. Consistent with this, treatment of C57Bl/6J mice with 10 mg/kg/day IMR-687 for 5 days displayed no effect on locomotor activity or classical fear conditioning (an animal model of learning and memory). In contrast, treatment with a brain penetrant PDE9i, developed for the treatment of neurologic diseases, significantly increased conditioned fear responses in these mice.ConclusionsIMR-687 increases HbF levels, and reduces red cell sickling, leukocytosis and microvascular stasis, without the observed toxicities of HU. IMR-687 may offer a once a day, oral, safe replacement for HU in the treatment of SCD. [Display omitted] DisclosuresMcArthur:Imara: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: Patent; Cydan Development: Employment, Equity Ownership. Maciel:Imara: Research Funding. Chen:Imara: Research Funding. Fricot:Imara: Research Funding. Kobayashi:Imara: Equity Ownership; Cydan Development: Employment. Nguyen:Imara: Research Funding. Parachova:Imara: Patents & Royalties. Abdulla:Imara: Research Funding. Vercellotti:Imara: Research Funding. Svenstrup:Imara: Consultancy, Patents & Royalties. Belcher:Imara: Research Funding; CSL-Behring: Research Funding.

Arginase Inhibition Reverses Endothelial Dysfunction, Pulmonary Hypertension, and Vascular Stiffness in Transgenic Sickle Cell Mice.

In sickle cell disease (SCD), hemolysis results in the release and activation of arginase, an enzyme that reciprocally regulates nitric oxide (NO) synthase activity and thus, NO production. Simply supplementing the common substrate L-arginine, however, fails to improve NO bioavailability. In this study, we tested the hypothesis that arginase inhibition would improve NO bioavailability and thereby attenuate systemic and pulmonary vascular endothelial dysfunction in transgenic mice with SCD. We studied 5-month-old transgenic sickle cell (SC) mice and age matched wild-type (WT) controls. SC mice were treated with the arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH; approximately 400 μg/d) for 4 weeks or left untreated. Vascular arginase activity was significantly higher at baseline in untreated SC mice compared to WT controls (SC versus WT, 346 ± 69.3 vs 69 ± 17.3 pmol urea/mg protein/minute; P = 0.0043; n = 4-5 animals per group). Treatment with ABH may significantly decrease arginase activity to levels near WT controls (SC + ABH 125.2 ± 17.3 pmol urea/mg protein/minute; P = 0.0213). Aortic strips from untreated SC mice showed decreased NO and increased reactive oxygen species (ROS) production (NO: fluorescence rate 0.76 ± 0.14 vs 1.34 ± 0.17 RFU/s; P = 0.0005 and ROS: fluorescence rate 3.96 ± 1.70 vs 1.63 ± 1.20 RFU/s, P = 0.0039; n = 3- animals per group). SC animals treated with ABH for 4 weeks demonstrated NO (fluorescence rate: 1.16 ± 0.16) and ROS (fluorescence rate: 2.02 ± 0.45) levels comparable with age-matched WT controls (n = 3- animals per group). The maximal endothelial-dependent vasorelaxation response to acetylcholine was impaired in aortic rings from SC mice compared with WT (57.7% ± 8.4% vs 80.3% ± 11.0%; P = 0.02; n = 6 animals per group). The endothelial-independent response was not different between groups. In SC mice, the right ventricular cardiac output index and end-systolic elastance were similar (4.60 ± 0.51 vs 2.9 ± 0.85 mL/min/100 g and 0.89 ± 0.48 vs 0.58 ± 0.11 mm Hg/μL), whereas the pulmonary vascular resistance index and right ventricular end-systolic pressure were greater (2.9 ± 0.28 vs 5.5 ± 2.0 mm Hg × min/μL/100 g and 18.9 ± 1.1 vs 23.1 ± 4.0 mm Hg; n = 8 animals per group). Pulse wave velocity (a measure of arterial stiffness) was greater in SC mice compared with WT (3.74 ± 0.54 vs 3.25 ± 0.21 m/s; n = 20 animals per group), arginase inhibition for 4 weeks significantly reduced the vascular SC phenotype to one similar to WT animals (P = 0.0009). Arginase inhibition improves NO bioavailability and thereby attenuates systemic and pulmonary vascular endothelial dysfunction in transgenic mice with SCD. Therefore, arginase is a potential therapeutic target in the treatment of cardiovascular dysfunction in SCD.

Abstract 347: Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Sickle Cell Disease (SCD) is characterized, in part, by vascular endothelial cell activation, increased oxidative stress and sickle cell adhesion. Excess levels of the vasoconstrictor, endothelin-1 (ET-1), activate endothelial cells, induce oxidative stress and inflammation in the vascular wall and regulate erythrocyte homeostasis, in part, via activation of protein disulfide isomerase (PDI). We measured circulating ET-1, TGFβ1 and PDI activity in two sickle transgenic knockout mouse models expressing human sickle hemoglobin (Hb), BERK and βS Antilles . We observed significant increases in all three markers (n=6; p&lt;0.01) when compared to either C57BL/J6 mice or knockout mice expressing normal human Hb A. We then intraperitoneally injected BERK mice for 14 days with ET-1 receptor antagonists and observed significant reductions in these circulating markers (n=6; p&lt;0.05). These studies are important as PDI is proposed to control hemostasis, thrombosis and is reported to regulate the major histocompatibility complex (MHC). In addition, activated endothelial cells express PDI and MHC. However, the relationship between ET-1 and MHC in SCD remains unclear. We characterized the role of ET-1 on MHC expression in the endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p&lt;0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p&lt;0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p&lt;0.05 n=4) in EA.hy926 cells. Increased expression of MHC was significantly blocked by co-incubation of cells with 10μM BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies showed that ET-1 incubation produced an increase in Histone H3 acetylation of the promoter in MHC molecules (HLA-A2 4.6 ± 1.4 folds, HLA-DRB 6.0 ± 0.7 folds, p&lt;0.01, n=3) in these cells; an event that was likewise blocked by BQ788. In addition, ET-1 stimulated recruitment of CIITA to the promoter of HLA-A2 (1.9 ± 0.7 folds, p&lt;0.05, n=3) and HLA-DRB (3.0 ± 0.6 folds, p&lt;0.01, n=3). These results implicate ET-1 as a novel regulator of MHC promoter activity and suggest that ET-1 receptor blockade represents an important therapeutic approach to improve both the inflammatory and vascular complications of SCD.

Flavonoid: Quercetin, a Multifunctional Therapeutic Agent to Reverse Acute Crisis in a Transgenic Sickle Mouse Model

Hypoxia acts as an initial trigger to amplify crisis episodes in SCD patients. The post capillary venular response to ischemia reperfusion injury includes abundant leukocyte adhesion, emigration, impairment of blood flow and enhanced oxidant production in transgenic sickle mice. Antioxidant mediated therapy to attenuate oxidative stress may reduce red cell sickling and inflammation and therefore may be beneficial for SCD. Based on this concept, we examined the efficacy of flavonoid quercetin to mitigate inflammation and vaso‐occlusion generated during hypoxia re‐oxygenation in acute sickle crisis model.MethodsAcute crisis in a transgenic sickle NY1DD mouse was experimentally induced by 18h hypoxia (8% oxygen, 0.5% CO2, and balance nitrogen) followed by 3h re‐oxygenation (H/R). NY1DD mice subjected to hypoxia were treated with a single dose of quercetin (200 mg/kg body weight) administered intraperitoneally at the onset of re‐oxygenation. In vivo intravital microscopic observation of cremaster post capillary venules was performed. Hemodynamic parameters were measured as described by Kaul et al 2004.ResultsNY1DD exhibits an intrinsic level of vaso‐occlusion and impairment of blood flow, which are increased during hypoxia re‐oxygenation. NY1DD mice exposed to H/R as indicated by an increased level of leukocyte adhesion (4.8± 0.58) and emigration (1.7± 0.28) vs normoxic NY1DD mice (1.6±0.26; 0.53±0.13). After treated with quercetin showed significant decreases in the level of leukocyte adhesion (0.47± 0.12; p&lt;0.0001) and emigration (0.22±0.09) vs untreated NY1DD and wild type (0.50 ±0.01; 0.16±0.05). Vaso‐occlusion, both intrinsic and induced by hypoxia in NY1DD mice was normalized completely to wild type after quercetin treatment. We also observed a significant increase in red cell velocity (2.8±0.24; p&lt;0.0001), wall shear rate (545±51.0; p&lt;0.0001) and flow rate (1.03±0.13; p&lt;0.001) after quercetin treatment when compared to untreated NY1DD (1.05±0.08; 211±17.8; 0.35±0.03) and wild type (3.5±0.3; 852.5±102.4; 1.25±0.17). All hemodynamic parameters were significantly increased after treatment with quercetin vs normoxia NY1DD, but didn't completely revert near to wild type. The level of lipid peroxidation product such as TBARS increased in H/R and reduced after quercetin treatment vs untreated NY1DD mice subjected to hypoxia. Expression of soluble cell adhesion molecules sP‐selectin, sE‐selectin and sVCAM‐1 were also significantly (p&lt;0.04; p&lt;0.02; p&lt;0.04) reduced in plasma after treated with quercetin vs untreated NY1DD mice. We also observed slight changes in red cell density pattern and reduction in high‐density red cells after quercetin treatment.ConclusionOur studies reveal that quercetin improves all hemodynamic parameters significantly and does not reverse back to wild type. Although, quercetin was completely attenuates vaso‐occlusion after hypoxia. These results suggesting need for further analysis of multiple doses of quercetin for longer period to verify whether hemodynamic parameters are being back to normal. We suggest that these effects may stems from the multifunctional properties of quercetin, which decreases sRBC interaction with vascular bed in the acute transgenic mice model. Our present data provide a strong basis for the therapeutic application of flavonoids in SCD. More studies are needed to better understand the mechanism of action of quercetin in vivo and to further establish its therapeutic effect in SCD.

Hepatic Overexpression of Hemopexin Inhibits Inflammation and Vascular Stasis in Murine Models of Sickle Cell Disease.

Sickle cell disease (SCD) patients have low serum hemopexin (Hpx) levels due to chronic hemolysis. We hypothesize that in SCD mice, hepatic overexpression of hemopexin will scavenge the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis. To examine the protective role of Hpx in SCD, we transplanted bone marrow from NY1DD SCD mice into Hpx™/™ or Hpx+/+ C57BL/6 mice. Dorsal skin fold chambers were implanted in week 13 post-transplant and microvascular stasis (% non-flowing venules) evaluated in response to heme infusion. Hpx™/™ sickle mice had significantly greater microvascular stasis in response to heme infusion than Hpx+/+ sickle mice (p<0.05), demonstrating the protective effect of Hpx in SCD. We utilized Sleeping Beauty (SB) transposon-mediated gene transfer to overexpress wild-type rat Hpx (wt-Hpx) in NY1DD and Townes-SS SCD mice. Control SCD mice were treated with lactated Ringer's solution (LRS) or a luciferase (Luc) plasmid. Plasma and hepatic Hpx were significantly increased compared to LRS and Luc controls. Microvascular stasis in response to heme infusion in NY1DD and Townes-SS mice overexpressing wt-Hpx had significantly less stasis than controls (p<0.05). Wt-Hpx overexpression markedly increased hepatic nuclear Nrf2 expression, HO-1 activity and protein, the heme-Hpx binding protein and scavenger receptor, CD91/LRP1 and decreased NF-κB activation. Two missense (ms)-Hpx SB-constructs that bound neither heme nor the Hpx receptor, CD91/LRP1, did not prevent heme-induced stasis. In conclusion, increasing Hpx levels in transgenic sickle mice via gene transfer activates the Nrf2/HO-1 anti-oxidant axis and ameliorates inflammation and vaso-occlusion.

Quantification of pain in sickle mice using facial expressions and body measurements

Pain is a hallmark feature of sickle cell disease (SCD). Subjects typically quantify pain by themselves, which can be biased by other factors leading to overtreatment or under-treatment. Reliable and accurate quantification of pain, in real time, might enable to provide appropriate levels of analgesic treatment. The mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability has been used to quantify varied types of pain. We hypothesized that addition of the objective parameters of body length and back curvature will strengthen the reproducibility of MGS. We examined MGS scores and body length and back curvature of transgenic BERK sickle and control mice following cold treatment or following treatment with analgesic cannabinoid CP55,940. We observed that sickle mice demonstrated decreased length and increased back curvature in response to cold. These observations correlate with changes in facial expression for the MGS score. CP55,940 treatment of sickle mice showed an increase in body length and a decrease in back curvature concordant with MGS scores indicative of an analgesic effect. Thus, body parameters combined with facial expressions may provide a quantifiable unbiased method for objective measure of pain in SCD.

Reduced Glutamate Channel Phosphorylation in Pain-Related Brain Areas in a Transgenic Model of Sickle Cell Disease

Sickle cell disease (SCD) is associated with chronic, debilitating pain beginning in childhood and persisting throughout life. SCD patients also tend to exhibit elevated rates of depression and anxiety disorders, indicating a possible connection between recurring pain episodes and affective or emotional dysregulation in this population. How SCD physiologically manifests within brain circuitry to mediate this link is currently unknown. In the current study, we utilized a transgenic mouse model of SCD to measure protein kinase A (PKA)-mediated phosphorylation of the AMPA glutamate receptor channel subunit GluR1 in specific brain regions associated with central nociception and the emotional dimension of pain, including the periaqueductal gray (PAG), prefrontal cortex (PFC), and hippocampus. Transgenic sickle mice (HbSS-BERK) expressing human sickle hemoglobin display significant mechanical and thermal hyperalgesia and exhibit reductions in GluR1 phosphorylation compared to littermate controls (HbAA-BERK) in the PAG, PFC, and hippocampus. This neuroadaptation is indicative of reduced excitatory transmission potential in these pain-associated areas. In comparison, GluR1 phosphorylation is unchanged in the dorsal striatum, a brain region not traditionally associated with pain or negative emotionality. Our data are consistent with other preclinical pain models that have linked chronic, peripheral pain conditions with compromised excitatory brain signaling. These results make a strong case for greater appreciation and investigation into mechanisms of possible affective disorder in individuals suffering from SCD. DisclosuresNo relevant conflicts of interest to declare.

Hemopexin Gene Therapy Inhibits Inflammation and Vaso-Occlusion in Transgenic Sickle Cell Mice

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). Hemolysis releases free hemoglobin (Hb) and Hb-containing microparticles into the vasculature that upon oxidation to methemoglobin frees heme from the globin, which in turn can promote oxidative stress and activate toll-like receptor 4 (TLR4) signaling. Hemopexin (HPX), a plasma β1-glycoprotein, binds heme with a very high affinity (Kd < 10-12 M), and transports it to the liver for catabolism via CD91 receptor-mediated uptake. SCD patients have low serum HPX levels likely due to chronic hemolysis leading to increased HPX catabolism with insufficient compensatory increase in synthesis. We and others have shown that HPX transports heme to the liver, and inhibits heme toxicity and the activation of endothelial, leukocyte and platelet TLR4 signaling. Acute studies have shown HPX infusion prior to a heme challenge protects sickle mice from vaso-occlusion and developing acute pulmonary injury while chronic HPX infusion therapy modified heme toxicity to endothelium. We hypothesize that in SCD mice, hepatic overexpression of HPX will bind the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis (vaso-occlusion). To examine the protective role of HPX in SCD, we transplanted bone marrow from NY1DD SCD mice into HPX-/- or normal C57BL/6 mice. After 12 weeks, conversion to the HbS phenotype was confirmed by isoelectric focusing. Dorsal skin fold chambers (DSFC) were implanted in week 13 and microvascular stasis (% non-flowing venules) assessed in response to heme (3.2 µmol/kg) infusion. HPX-/- sickle mice had 34% ± 3% and 24% ± 2% at 1h and 4h post heme, significantly greater than HPX+/+ C57BL/6 sickle mice which had 21% ± 5% and 13% ± 8% at 1 and 4 h, (mean ± SD, p<.05), demonstrating the protective role of HPX in SCD. To further test our hypothesis, we utilized Sleeping Beauty (SB) transposon-mediated gene therapy to overexpress rat HPX in NY1DD and Townes-SS SCD mice. Rat HPX plasmid (pT2/Caggs-HPX) was delivered with an SB transposase plasmid (pK/CMV-SB 100X) and luciferase (LUC) plasmid (pT2/Caggs-LUC, as tracer) in trans into NY1DD or Townes-SS SCD mice by hydrodynamic tail vein injections. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or LUC plasmid with SB transposase plasmid in trans. One week later, the mice LUC bioluminescence imaging showed the liver was the primary location of expression. Four weeks later, the HPX SCD mice had marked increases in hepatic rat HPX mRNA (300-2000 copies/5ng total RNA) comparing to LRS and SB-LUC controls (0-44 copies/5ng total RNA). Plasma and hepatic HPX were significantly increased compared to LRS and SB-LUC controls. In vitro expression of the rat HPX plasmid in Chinese Hamster Ovary cells, and protein purification confirmed heme binding activity by spectroscopic scan absorbance shifts of rat HPX-heme complexes at 414nm.DSFCs were implanted 4 weeks after plasmid infusion and microvascular stasis was assessed in response to heme (3.2 µmol/kg) infusion. NY1DD and Townes-SS mice overexpressing rat HPX (SB-HPX) had significantly less stasis than LRS or SB-LUC treated SCD mice (Figure 1A and B). HPX overexpression markedly increased nuclear Nrf2 expression in the livers of sickle mice, presumably by promoting delivery of heme to the liver and activating the Keap1-Nrf2 axis. In addition, hepatic HO-1 activity and protein and CD91 protein were increased in sickle mice overexpressing HPX and NF-ĸB activation was markedly decreased as assessed by nuclear phospho-p65-NF-ĸB expression on western blots demonstrating the anti-inflammatory properties of HPX in sickle mice. In conclusion, supplementing HPX levels in transgenic sickle mice via gene therapy activates the Nrf2 anti-oxidant axis and ameliorates inflammation and vaso-occlusion. We speculate that plasma HPX supplementation may be beneficial in SCD especially during hemolytic crises or acute chest syndrome. [Display omitted] DisclosuresVercellotti:Cydan: Research Funding; CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Belcher:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding.

Oxidative Stress Contributes to Overactive Bladder in the Transgenic Sickle Cell Mouse

Introduction: Lower urinary tract symptoms (LUTS) such as nocturia, urinary frequency, and urgency have been clinically reported in children and adults with sickle cell disease (SCD; Portocarrero et al J Urol 2012; Anele et al Neurourol Urodyn 2015), but little is known about the pathophysiology of micturition dysfunction in SCD patients. Transgenic sickle cell mice have been employed to better understand SCD pathophysiology. Recently, we reported that Berkeley SCD mice display underactive bladder and reduced detrusor smooth muscle contractions (Claudino et al Plos One, 2015). The homozygous Townes transgenic sickle cell mouse is another model for SCD, but urinary bladder dysfunction has never been studied in this model. Considering that SCD is associated with elevated ROS production, we hypothesized that increased ROS levels lead to alterations in the urinary bladder function of Townes SCD mice. Thus, the aim of this study was to evaluate the micturition and detrusor smooth muscle contractions in Townes transgenic sickle cell mice.Methods: Townes transgenic sickle cell mice and C57BL/6 mice (control) aged 3 to 4 months old were used. Cystometry was performed to evaluate the urinary function in vivo. In separate protocols, the urinary bladder was removed and placed in Krebs solution. Two longitudinal detrusor smooth muscle strips with intact urothelium were obtained from each bladder. The strips were mounted in 4-ml organ baths containing Krebs-Henseleit solution at 37°C continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4). Cumulative concentration-response curves to muscarinic receptor agonist carbachol (CCh; 0.01-100 μM), chloride potassium (KCl; 1-300mM) and frequency-response curves for electrical field stimulation (EFS; 1-32Hz) were obtained in strips of detrusor smooth muscle from both control and SCD mice.mRNA expression for gp91phox and SOD-1 in bladder were also evaluated.Results: Micturition in SCD mice was irregular, and characterized by significant (P < 0.05) increases in the frequency of voiding contractions and nonvoiding contractions (n=5). Voiding pressure, basal pressure, capacity and threshold pressure were not changed in SCD mice. Carbachol (0.01 - 100 µM) induced concentration-dependent detrusor smooth muscle contractions in both control and SCD mice, but maximal contractile responses were significantly lower (P < 0.05) in SCD compared to control mice (1.40 ± 0.4 and 2.9 ± 0.2 mN/mg, respectively; n=5). Likewise, EFS-induced neurogenic detrusor contractions in SCD mice were 29% lower (P < 0.05) compared to the control (n=10). Contractile responses induced by KCl (1 - 300 mM) did not differ between control and SCD group (n=5). The mRNA expression for the NADPH subunit gp91phox in bladder was about 2-fold higher (P<0.05) in SCD mice compared to the control group, whereas no changes in the mRNA expression for SOD-1 were observed among groups (n=6). Infiltration by inflammatory cells (mononuclear cells) was seen in the submucosa and muscular area of bladder from SCD mice (n=4).Conclusion: Our study shows that the overactive bladder seen in SCD mouse is associated with upregulation of NADPH oxidase subunit gp91phox. Interestingly, in the contrast, the Berkeley SCD mouse exhibits an atonic detrusor smooth muscle and underactive bladder (Claudino et al Plos One 2015). One may speculate therefore that the overactive bladder in Townes SCD may progress to underactive bladder at latter stages. To confirm this hypothesis, we will evaluate the urinary bladder function in older SCD mice.Financial Support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Moderate Exercise Training Attenuates Inflammation in Transgenic Sickle Cell Mice

Sickle cell disease (SCD) is a monogenic disorder characterized by severe systemic and vascular inflammation. There is strong evidence that moderate exercise training reduces inflammation in healthy people as well as in multiple diseases conditions. The aim of this study was to characterize the effects of moderate exercise training in a humanized transgenic mouse model of SCD (Townes) in mice expressing exclusively human sickle hemoglobin (SS). The SS mice display many of the features of human SCD including systemic inflammation and chronic anemia. We designed a treadmill training protocol of 1hr/day, 5 days a week for 8 weeks. Prior to the training, the maximal oxygen uptake (V̇O2max) was assessed by an exhaustive incremental exercise. SS mice (n=22) had lower V̇O2max (mean ± SEM: 7,068 ml/kg/hr ± 133 vs. 8,407 ml/kg/hr ± 224.5, p<0.0001) and lower maximal speed (14.3 m/min ± 0.7 vs. 29.3 m/min ± 1.7, p<0.0001) than control AA mice expressing exclusively normal human hemoglobin (n=12). Together these results indicate SS mice have an inherently reduced aerobic exercise capacity; nonetheless, they tolerated a training regimen at 60% of V̇O2max. After eight weeks of training, SS mice had a lower hematocrit (20.3% ± 0.8% vs. 24.4% ± 1.8%, p=0.04; n=8) than untrained SS littermates that were exposed to all other experimental conditions except training. There were only moderate changes in red blood cell count (Trained: 4.8.106/µL ± 0.2, Untrained: 5.7.106/µL ± 0.5, p>0.05) or total hemoglobin (Trained: 5.6 g/dL ± 0.2 vs 6.3 g/dL ± 0.4, p>0.05) between the two groups suggesting the low hematocrit in the trained mice was probably due to increased plasma volume, which is a normal physiological response to exercise training. The spleen/body mass ratio was significantly lower in the trained SS mice compared to the untrained littermates (5.7 mg/g body weight ± 0.3 vs. 7.0 mg/g body weight ± 0.9, p=0.04). Importantly, the spleen was less congested in the trained SS mice, and the magnitude of congestion in all animals (trained and untrained) correlated very strongly with the relative spleen mass (r=0.7, p=0.009). Exercise training significantly increased the percent of oxyhemoglobin in the venous blood of SS mice (Trained: 39% ± 4.3, Untrained: 22.3 ± 4.1, p=0.016). And, there was a corresponding decrease in the percent carboxyhemoglobin (Trained: 2.3% ± 0.7, Untrained 6.2% ± 0.5, p=0.001). These data suggest moderate exercise training may reduce the proportion HbS that is deoxygenated in the venous circulation. In addition, exercise training significantly reduced the peripheral blood white cell count in the SS mice (Training: 14.7.103/µL ± 0.9, Untrained: 21.6.103/µL ± 3.1, p=0.03); this change was reflected by a corresponding lower lymphocyte count in the trained mice (12.103/µL ± 0.8 vs. 17.3.103/µL ± 2.3, p=0.028). Finally, running decreased the liver expression of heme oxygenase-1 (HO-1) mRNA (p=0.04) and the plasma concentration of macrophage inflammatory protein-1β (MIP-1β, p=0.03). Interestingly, HO-1 is implicated in metabolic inflammation and MIP-1β promotes endothelial adhesion of CD8+ T cell through a VCAM-1 dependent pathway. Together with the reductions in leukocyte counts, we suggest that moderate exercise training may reduce sequestration of sickle erythrocytes/congestion, resting blood deoxygenation and inflammation in SCD. DisclosuresNo relevant conflicts of interest to declare.

Protein Disulfide Isomerase in Sickle Cell Disease

Protein disulfide isomerase (PDI), a multifunctional oxidoreductase that regulates thrombus formation, leukocyte adherence to the endothelium and nitric oxide delivery, is present at high levels and regulates KCNN4 (Gardos Channel) activity in erythrocytes from humans with Sickle Cell Disease (SCD) and sickle mouse models. We reported evidence of elevated erythrocyte-surface associated and circulating PDI activity in humans and mouse models of SCD when compared to either wild-type mice, transgenic mice expressing normal human globins or otherwise healthy individuals; results that suggest a novel role for PDI in the pathophysiology of SCD through unclear mechanisms. We studied the effects of deoxygenation (5% O2) on PDI activity in endothelial cells and sickle erythrocytes from humans with SCD and two sickle transgenic knockout mouse models expressing human sickle hemoglobin, BERK and βSAntilles. Our data shows that deoxygenation of human and mouse sickle erythrocytes increased surface-associated reductive capacity that was sensitive to monoclonal antibodies against PDI (mAb PDI [RL-90]). We then studied sickle human erythrocytes and showed that PDI inhibitors (quercentin-3-rutinoside [Q3R] and mAb PDI) significantly reduced deoxy-stimulated sickle cell dehydration and Gardos channel activity (n=5; P <0.03). We characterized erythrocyte density with a phthalate density-distribution assay, generated density distribution curves and calculated the D50. Both mAb PDI and Q3R significantly reduced D50 when compared to vehicle (1.113±0.002 to 1.102±0.001 [P <0.0001]; and 1.101±0.002 [P <0.0001]; respectively). In contrast, incubation with exogenous PDI (3 nM) increased cellular dehydration (from D50= 1.110 ± 0.001 to D50= 1.115 ±0.001, P <0.01). We also measured the effect of hypoxia and endothelin-1 (ET-1; 10nM) in the human vascular endothelial cell line, EA.hy926. We observed that hypoxia induced PDI secretion that was further enhanced by co-incubation with ET-1 (10nM; n=3; P <0.05). The selective inhibitor of ET-1 subtype B receptor antagonist, BQ788 blocked ET-1 stimulated PDI increases in these cells as was previously reported in red blood cells. Consistent with these results hypoxia was associated with increased mRNA expression of MCP-1, VEGF-a but not ICAM by qRT-PCR and Taqman probes (n=3; P <0.05). Of importance we observed that early cultures of mouse aortic endothelial cells from BERK mice showed similar results. We then evaluated the effects of PDI on erythrocyte hemolysis by exposing cells from patients with SCD to 20 mM 2-2'-azo-bis- (2-amidinopropane) dihydrochloride (AAPH) with or without Q3R or mAb PDI. AAPH-induced hemolysis was dose-dependently blocked by Q3R (IC50: 4.1nM; n=6, P <0.0001 compared to vehicle) or mAb PDI (IC50: 11nM; n=3). Irrelevant IgG did not affect hemolysis under these conditions. Experiments performed in blood from BERK or βSAntilles mice showed similar results. We then studied sickle mice that express varying levels of HbF; BERK (<1% HbF), BERKγM (25% HbF), and BERKγH (45% HbF). BERKγH had the lowest circulating and cell associated PDI activity among the three mouse types that was associated with lower circulating ET-1 levels (n=2; P <0.05). Consistent with these results we observed an inverse correlation between levels of HbF and PDI activity in cells from humans with SCD (n=4). Thus we posit that in SCD elevated erythrocyte PDI activity is important for cell survival and stability and that its inhibition may represent a novel therapeutic approach for improving both the hematological and vascular complications of SCD as it may not only increase sickle erythrocyte survival but may likewise interfere with cellular adhesion leading to reduced vaso-occlusive episodes. [Supported by NIH: HL090632 (AR) and HL096518 (JRR)] DisclosuresNo relevant conflicts of interest to declare.

Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice.

BackgroundUrological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking.ObjectiveThus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS).MethodsUrine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1nM-10μM) and contractile response to (carbachol (CCh; 1 nM-100μM), KCl (1 mM-300mM), CaCl2 (1μM-100mM), α,β-methylene ATP (1, 3 and 10 μM) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100μM) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder.ResultsSS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective β3-adrenergic agonist (mirabegron) and to a non-selective β-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, α, β-methylene ATP, KCl, extracellular Ca2+ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration.ConclusionsTaken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients.

Mechanism of testosterone deficiency in the transgenic sickle cell mouse.

Testosterone deficiency is associated with sickle cell disease (SCD), but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle) exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH) levels compared with wild type (WT) mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol)- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR), but not cholesterol side-chain cleavage enzyme (P450scc), in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi) exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

Chronic Physical Activity In Transgenic Sad Mice

Sickle cell anaemia (SCA) is characterized by hemorheological disorders, enhanced oxidative stress and impaired vascular function which promote the risk to trigger acute painful vaso-occlusive crises (VOC). The finding of new therapeutic approaches to improve the biological profile and the clinical management of SCA patients is a permanent challenge. As for cardiac and metabolic diseases, individualized chronic exercise programs have been suggested to improve the health status in SCA but until now no study tested this hypothesis in humans because of the vaso-occlusive risks that SCA patients could encounter during such a protocol. PURPOSE: To determine if 8 weeks of voluntary wheel running may improve blood rheology and nitric oxide bioavailability (NOx) and reduce oxidative stress in transgenic sickle mice model. METHODS: C57Bl/6J (C57) and transgenic SAD mice (31.7 ±3.6 weeks old) were randomly divided in sedentary (SED) or physical activity (PA) group. C57PA and SADPA were housed in cages equipped with running wheels. After 8 weeks, mice were euthanized either after normoxic conditions or hypoxia/reoxygenation (H/R) stress (4 hours at 6.5% oxygen followed by 2 hours of reoxygenation in ambient air). Blood samples were used to measure hematocrit and blood viscosity at two shear rates (18.75 and 375 s-1) and hematocrit-to-blood viscosity ratio (HVR) was calculated as the ratio of both values. Oxidative stress markers (malondialdehyde, MDA) and antioxidant enzymes (glutathione peroxidase, GPX; superoxide dismutase, SOD, catalase) were assayed on the heart and NOx levels were determined in the lungs using nitrite/nitrate assay. RESULTS: Chronic exercise training decreased blood viscosity (-18.2%, P<0.05), increased HVR ratio (+40%, P<0.01) at moderate shear rate but not at high shear rate and enhanced enzymatic antioxidant activity of GPX (+96%, P<0.05) in normoxia in SAD mice. Simultaneously in response to H/R stress MDA and NOx concentrations respectively decreased (-22.4%, P<0.05) and increased (P<0.05) in SADPA mice compared with SADSED. CONCLUSION: Physical activity in SAD mice seems to be an efficient way to reduce biological factors involved in VOC events via the improvement of microvascular blood flow, redox status and NOx bioavailability.

Body composition and grip strength are improved in transgenic sickle mice fed a high-protein diet.

Key pathophysiology of sickle cell anaemia includes compensatory erythropoiesis, vascular injury and chronic inflammation, which divert amino acids from tissue deposition for growth/weight gain and muscle formation. We hypothesised that sickle mice maintained on an isoenergetic diet with a high percentage of energy derived from protein (35 %), as opposed to a standard diet with 20 % of energy derived from protein, would improve body composition, bone mass and grip strength. Male Berkeley transgenic sickle mice (S; n 8-12) were fed either 20 % (S20) or 35 % (S35) diets for 3 months. Grip strength (BIOSEB meter) and body composition (dual-energy X-ray absorptiometry scan) were measured. After 3 months, control mice had the highest bone mineral density (BMD) and bone mineral content (BMC) (P<0·005). S35 mice had the largest increase in grip strength. A two-way ANOVA of change in grip strength (P=0·043) attributed this difference to genotype (P=0·025) and a trend in type of diet (P=0·067). l-Arginine (l-Arg) supplementation of the 20 % diet was explored, as a possible mechanism for improvement obtained with the 35 % diet. Townes transgenic sickle mice (TS; n 6-9) received 0·8, 1·6, 3·2 or 6·4 % l-Arg based on the same protocol and outcome measures used for the S mice. TS mice fed 1·6 % l-Arg for 3 months (TS1.6) had the highest weight gain, BMD, BMC and lean body mass compared with other groups. TS3.2 mice showed significantly more improvement in grip strength than TS0·8 and TS1.6 mice (P<0·05). In conclusion, the high-protein diet improved body composition and grip strength. Outcomes observed with TS1.6 and TS3.2 mice, respectively, confirm the hypothesis and reveal l-Arg as part of the mechanism.