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

Development of cellular and gene therapies for b[beta]-Thalassemia and sickle cell disease

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The Interplay between Cyclic AMP, MAPK, and NF-κB Pathways in Response to Proinflammatory Signals in Microglia

Cyclic AMP is an important intracellular regulator of microglial cell homeostasis and its negative perturbation through proinflammatory signaling results in microglial cell activation. Though cytokines, TNF-α and IL-1β, decrease intracellular cyclic AMP, the mechanism by which this occurs is poorly understood. The current study examined which signaling pathways are responsible for decreasing cyclic AMP in microglia following TNF-α stimulation and sought to identify the role cyclic AMP plays in regulating these pathways. In EOC2 microglia, TNF-α produced a dramatic reduction in cyclic AMP and increased cyclic AMP-dependent PDE activity that could be antagonized by Rolipram, myristoylated-PKI, PD98059, or JSH-23, implicating a role for PDE4, PKA, MEK, and NF-κB in this regulation. Following TNF-α there were significant increases in iNOS and COX-2 immunoreactivity, phosphorylated ERK1/2 and NF-κB-p65, IκB degradation, and NF-κB p65 nuclear translocation, which were reduced in the presence of high levels of cyclic AMP, indicating that reductions in cyclic AMP during cytokine stimulation are important for removing its inhibitory action on NF-κB activation and subsequent proinflammatory gene expression. Further elucidation of the signaling crosstalk involved in decreasing cyclic AMP in response to inflammatory signals may provide novel therapeutic targets for modulating microglial cell activation during neurological injury and disease

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

Histological analysis of elastin content in media (A) and representative microscopy images of Verhoeff van Gieson-stained sections (B) of the left common carotid arteries in control (open bars) and homo-βthal mice (closed bars, <i>n</i> = 8 per group). Data are means ± SEM.

<p>^*<i>p</i><0.05. L, vascular lumen.</p

Comparison of structural characteristics in common carotid arteries from control (○; <i>n</i> = 13) and homo-βthal mice (•; <i>n</i> = 11).

<p>(A) Luminal diameter, (B) external diameter, (C) wall cross-sectional area (CSA), and (D) wall-to-lumen ratio versus intraluminal pressure. Data are means ± SEM. ^†<i>p</i><0.001.</p

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

Hematologic parameters.

<p>Values are means±SEM. ^*<i>p</i><0.01; ^†<i>p</i><0.001 vs. control mice. RBC, red blood cell count; Hb, hemoglobin; Hct, hematocrit; MCV, mean RBC cellular volume; MCH, mean RBC cellular hemoglobin; Retics, Reticulocytes.</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