Vascular Endothelial Dysfunction in β-Thalassemia Occurs Despite Increased eNOS Expression and Preserved Vascular Smooth Muscle Cell Reactivity to NO

The hereditary β-thalassemia major condition requires regular lifelong blood transfusions. Transfusion-related iron overloading has been associated with the onset of cardiovascular complications, including cardiac dysfunction and vascular anomalies. By using an untransfused murine model of β-thalassemia major, we tested the hypothesis that vascular endothelial dysfunction, alterations of arterial structure and of its mechanical properties would occur despite the absence of treatments.Vascular function and structure were evaluated ex vivo. Compared to the controls, endothelium-dependent vasodilation with acetylcholine was blunted in mesenteric resistance arteries of β-thalassemic mice while the endothelium-independent vasodilator (sodium nitroprusside) produced comparable vessel dilation, indicating endothelial cell impairment with preserved smooth muscle cell reactivity to nitric oxide (NO). While these findings suggest a decrease in NO bioavailability, Western blotting showed heightened expression of aortic endothelial NO synthase (eNOS) in β-thalassemia. Vascular remodeling of the common carotid arteries revealed increased medial elastin content. Under isobaric conditions, the carotid arteries of β-thalassemic mice exhibited decreased wall stress and softening due to structural changes of the vessel wall.A complex vasculopathy was identified in untransfused β-thalassemic mice characterized by altered carotid artery structure and endothelial dysfunction of resistance arterioles, likely attributable to reduced NO bioavailability despite enhanced vascular eNOS expression

<i>Gfi1b</i> deficiency causes delayed and incomplete silencing of embryonic globin genes.

<p>A: Q-PCR analysis of embryonic globin gene and <i>Gata1</i> expression in CD71⁺, TER119⁻ and CD71⁺, TER119⁺ fetal liver cells from wild type and <i>EpoR</i>-Cre induced <i>Gfi1b</i> knockout embryos at 15.5 dpc. Bar graphs show the fold change of expression of the indicated genes in <i>Gfi1b</i> knockout cells over the wild type expression. Error bars indicate the standard deviation of at least three replicates. B: Line graphs represent the results of the Q-PCR analysis of globin gene expression in fetal liver cells of <i>EpoR</i>-Cre, <i>Gfi1b</i>^fl/fl (<i>Gfi1b</i> KO) or wild type (WT) mice during developmental stages from 10.5 dpc to 16.5 dpc depicted as percent of total globin (logarithmic scale). Error bars represent the standard deviation from triplicate measurements from two (10.5 and 13.5 dpc) to four (12.5, 14.5, 15.5, 16.5 dpc) individuals of each genotype. C: Bar graph representing the results of triplicate Q-PCR analysis of the expression of globin genes, Gata-1 and -2 in <i>Gfi1b</i>^GFP/GFP homozygous <i>Gfi1b</i> deficient mice compared to heterozygous and wild type littermates at 13.5 dpc. Numbers above bars are fold changes calculated as 2^{(Δct KO – Δct WT)}.</p

Ablation of <i>Gfi1b</i> in adult <i>Rosa</i>-Cre-ERT, <i>Gfi1b</i>^GFP/fl mice by tamoxifen confirms defects in erythroid cell fate decision and maturation causing compensated anemia.

<p>A: Protocol used to efficiently generate adult <i>Gfi1b</i>-KO mice using tamoxifen. Eight week old <i>Gfi1b</i>^fl/GFP mice either carrying a Cre-ERT knock-in into the <i>Rosa</i>26 locus (inducible <i>Gfi1b</i>-KO) or not (wt control) were treated by gavage with tamoxifen (100 mg/kg body weight) freshly dissolved in corn-oil at day 0, 1, 2 and 5 and sacrificed for analysis at day 14 of the experiment. B: Flow cytometric analysis of total bone marrow cells or FACS sorted Lin⁻, cKit⁺ cells from wild type and <i>Gfi1b</i>-KO mice for the presence of the indicated markers (TER119, CD71, CD34 and CD16/32. C: Flow cytometric analysis of stress erythropoiesis in the spleen using cells from wild type (left panel) or <i>Gfi1b</i>-KO (right panel) mice. FACS plots from (B) and (C) are representative for three or more individual samples from each genotype.</p

<i>Gfi1b</i> regulates maturation of erythroid cells in adult mice.

<p>A: <i>Gfi1b</i>^fl/fl mice either carrying (<i>Gfi1b</i>-KO) the <i>Mx</i>-Cre transgene or not (wild type) were treated five times with pIpC (500 µg each) every other day and sacrificed for analysis 42 days after the first injection. B: Flow cytometric analysis of progenitors (middle panel) and maturing erythrocytes from the bone marrow (upper and lower panel) and spleen (lower panel) of wild type and <i>Gfi1b</i>-KO mice. TER119⁺ bone marrow cells from pIpC induced wild type and <i>Gfi1b</i> knockout animals were isolated by flow cytometry and RNA was prepared for microarray analysis of gene expression as indicated (lower left panel). FACS plots are representative for at least four individual samples from each genotype. C: Peripheral blood of wild type and conditional <i>Gfi1b</i>-KO mice was analyzed using an ADVIA hematology system and the comparison results are presented as box-whisker plots showing the central location and distribution of the indicated measures. Red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), macrocytic RBCs (Macro), mean corpuscular volume (MCV), reticulocytes (Retic), red cell size and shape (RDW), immature reticulocytes fraction high (IRF-H) and white blood cell count (WBC).</p

Evidence for a Novel Mechanism Independent of Myocardial Iron in β-Thalassemia Cardiac Pathogenesis

<div><p>Human β-thalassemia major is one of the most prevalent genetic diseases characterized by decrease/absence of β-globin chain production with reduction of erythrocyte number. The main cause of death of treated β-thalassemia major patients with chronic blood transfusion is early cardiac complications that have been attributed to secondary iron overload despite optimal chelation. Herein, we investigated pathophysiological mechanisms of cardiovascular dysfunction in a severe murine model of β-thalassemia from 6 to 15-months of age in the absence of confounding effects related to transfusion. Our longitudinal echocardiography analysis showed that β-thalassemic mice first display a significant increase of cardiac output in response to limited oxygen-carrying erythrocytes that progressed rapidly to left ventricular hypertrophy and structural remodeling. Following this compensated hypertrophy, β-thalassemic mice developed age-dependent deterioration of left ventricular contractility and dysfunction that led toward decompensated heart failure. Consistently, murine β-thalassemic hearts histopathology revealed cardiac remodeling with increased interstitial fibrosis but virtual absence of myocardial iron deposits. Importantly, development of thalassemic cardiac hypertrophy and dysfunction independently of iron overload has uncoupled these cardiopathogenic processes. Altogether our study on β-thalassemia major hemoglobinopathy points to two successive phases resulting from severe chronic anemia and from secondarily induced mechanisms as pathophysiologic contributors to thalassemic cardiopathy.</p> </div

Endothelium-dependent vasodilatory responses of mesenteric resistance arterioles from control (<i>n</i> = 10) (A) and homo-βthal mice (<i>n</i> = 9) (B) to acetylcholine (ACh) in the absence (○) or presence (

<p>▴<b>) of L-NAME.</b> Relaxation responses are expressed as a percentage increase in lumen diameter after norepinephrine pre-contraction. Data are means ± SEM. ^*<i>p</i><0.05 and ^†<i>p</i><0.001.</p

<i>Growth Factor Independence 1b</i> (<i>Gfi1b</i>) Is Important for the Maturation of Erythroid Cells and the Regulation of Embryonic Globin Expression

<div><p>Growth factor independence 1b (GFI1B) is a DNA binding repressor of transcription with vital functions in hematopoiesis. <i>Gfi1b</i>-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of <i>Gfi1b</i>, we used conditionally deficient mice that harbor floxed <i>Gfi1b</i> alleles and inducible (<i>Mx</i>-Cre, Cre-ERT) or erythroid specific (<i>EpoR</i>-Cre) Cre expressing transgenes. In contrast to the germline knockout, <i>EpoR</i>-Cre mediated erythroid specific ablation of <i>Gfi1b</i> allows full gestation, but causes perinatal lethality with very few mice surviving to adulthood. Both the embryonic deletion of <i>Gfi1b</i> by <i>EpoR</i>-Cre and the deletion in adult mice by <i>Mx</i>-Cre or Cre-ERT leads to reduced numbers of erythroid precursors, perturbed and delayed erythroid maturation, anemia and extramedullary erythropoiesis. Global expression analyses showed that the <i>Hba-x</i>, <i>Hbb-bh1</i> and <i>Hbb-y</i> embryonic globin genes were upregulated in <i>Gfi1b</i> deficient TER119⁺ fetal liver cells over the gestation period from day 12.5–17.5 p.c. and an increased level of <i>Hbb-bh1</i> and <i>Hbb-y</i> embryonic globin gene expression was even maintained in adult <i>Gfi1b</i> deficient mice. While the expression of <i>Bcl11a</i>, a regulator of embryonic globin expression was not affected by <i>Gfi1b</i> deficiency, the expression of <i>Gata1</i> was reduced and the expression of <i>Sox6</i>, also involved in globin switch, was almost entirely lost when <i>Gfi1b</i> was absent. These findings establish <i>Gfi1b</i> as a regulator of embryonic globin expression and embryonic and adult erythroid maturation.</p></div

Longitudinal analysis of body weight in homo-βthal mice at 6, 10 and 14 months.

<p>Homo-βthal mice (filled bars) have significantly decreased body weight relative to controls (open bars) at 10 and 14 months (^*<i>p</i><0.05, ^**<i>p</i><0.01). Within the control group, body weight was increased at 10 and 14 months vs 6 months (^a<i>p</i><0.05, ^b<i>p</i><0.001), and 14 months vs 10 months (^c<i>p</i><0.05). Values are means±SEM and analyzed by two-way ANOVA.</p

Insufficient activation of <i>Sox6</i>, <i>Gata1</i> and <i>Gpa</i> in <i>Gfi1b</i> deficient cells.

<p>A: Q-PCR analysis of the relative expression levels of regulators of globin gene expression (<i>Gata1</i>), globin gene switch (<i>Blc11a, Sox6</i>) and glycophorin A (<i>Gypa</i>) normalized to <i>Gapdh</i>. Sample sizes were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096636#pone-0096636-g007" target="_blank">Figure 7B</a>. B: Q-PCR analysis on RNA from CD71⁺, TER119⁻ (pro-erythroblasts) and TER119⁺ (late erythroblasts) live bone marrow cells from a surviving <i>EpoR</i>-Cre induced <i>Gfi1b</i>-KO mouse compared to a wild type littermate. All measurements were done in triplicates. C: RT-PCR detection of <i>EpoR</i>-Cre and <i>Gfi1b</i> wt, flox and KO (excised) alleles on total RNA from bone marrow of a surviving mouse with erythroid specific inactivation of <i>Gfi1b</i> by <i>EpoR</i>-Cre.</p

Characteristics of 14-month-old mice.

<p>Values are means ± SEM.</p>*<p><i>p</i><0.05;</p>†<p><i>p</i><0.001 vs. control mice. BW, body weight; Hct, hematocrit.</p