Hady Felfly

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

Aims 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. Methods and Results 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. Conclusions 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.

Alternative treatment paradigm for thalassemia using iron chelators.

OBJECTIVE beta-thalassemia major, or Cooleys anemia, is a red blood cell disorder requiring lifelong blood transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction in high numbers. We hypothesized that removing this proximate iron compartment as a primary treatment, using standard and alternative orally active iron chelators, could prevent hastened red cell removal and, clinically, perhaps alleviate the need for transfusion. MATERIALS AND METHODS Iron chelators of the pyridoxal isonicotinoyl hydrazone family (pyridoxal isonicotinoyl hydrazone and its analog pyridoxal ortho-chlorobenzoyl hydrazone) were evaluated in addition to the present mainstay, desferrioxamine and deferiprone, in vitro and in vivo. RESULTS Treatment of human beta-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron and reduction of oxidative stress, as evaluated by methemoglobin levels. When administered to beta-thalassemic mice, iron chelators mobilized erythrocyte membrane iron, reduced cellular oxidation, and prolonged erythrocyte half-life. The treated thalassemic mice also showed improved hematological abnormalities. Remarkably, a beneficial effect as early as the erythroid precursor stage was manifested by normalized proportions of mature vs immature reticulocytes. All four compounds were also found to mitigate iron accumulation in target organs, a critical determinant for patient survival. In this respect, pyridoxal ortho-chlorobenzoyl hydrazone displayed higher activity relative to other chelators tested, further diminishing iron in liver and spleen by up to approximately fivefold and twofold, respectively. CONCLUSION Our study demonstrates the ability of iron chelators to improve several of the fundamental pathological disturbances of thalassemia, and reveals their potential for clinical use in diminishing requirement for transfusion when administered early in disease development.

Long-term correction of beta-thalassemia with minimal cellular requirement and transplantation modalities.

Determination of minimal criteria, pre-transplantation regimens, and infusion modalities for effective and reproducible bone marrow (BM) therapy in β-thalassemia is of fundamental importance for clinical application. In this study, using repopulation assays, we first established the minimal proportion of normal BM stem cells that would result in therapeutic benefit in this red blood cell (RBC) disorder. Eight groups of stable chimeric hemizygous β-thalassemic (hemi-βthal) mice (10-89%) were systematically subjected to cellular, molecular, and patho-physiologic analyses for ∼2 years. In the chimeric hemi-βthal groups containing 19-24% normal donor cells, all RBC parameters and consequent erythropoiesis were significantly improved. Mice in the 24% chimeric group and above had marked reduction in organ pathology including iron deposits, and survived to a normal lifespan. Altogether, these results established that a range of 19-24% normal BM cells is sufficient for long-term significant correction of the hemi-βthal phenotype. We also determined concomitantly the minimal myelosuppression radiation doses, the number of cells to be infused, and the number of infusions required in order to attain this therapeutic range in hemi-βthal mice. Importantly, with prior minimal myelosuppression with 1 or 2 Gy, and using cell doses of 40 or 60 millions, 100% of the recipients were successfully engrafted at therapeutic levels, provided the cells were administered in two doses. This study has therefore determined the therapeutic chimeric level as 19-24% of normal cells, and has also defined the minimal transplantation modalities necessary for the stable and successful correction of the hemi-βthal phenotype.

Evidence for a novel mechanism independent of myocardial iron in β-thalassemia cardiac pathogenesis.

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.

Successful correction of murine sickle cell disease with reduced stem cell requirements reinforced by fractionated marrow infusions

Minimal criteria requirements of stem cell replacement, conditioning regimen and modalities of infusion essential for cure of sickle cell disease (SCD) by bone marrow(BM)/stem cell transplantation or gene therapy must be established prior to clinical trials. The threshold of normal BM/stem cells for therapeutic correction of this red blood cell disorder was evaluated in the SAD murine SCD model from peripheral donor white blood cells. From 11 groups of stable chimeric SAD mice (5–92%) analyzed over ∼2 years, mice with ∼16% normal donor stem cells showed improvement of haematological and erythroid responses. Mice in the 26% chimeric group and above demonstrated substantial amelioration of organ pathologies with generalized decreased iron deposits, fibrosis and reached normal lifespan. Subsequently, the minimal myelosuppression concurrently with number and timing of infusions and number of BM cells was determined to reach therapeutic threshold in SAD mice. Higher myelosuppression (2 Gy vs. 1 Gy) and cell number in single infusion led to increased chimerism. Importantly, administration of three‐equivalent cell subdoses within 28 h of mild myelosuppression resulted in 100% recipient engraftment at therapeutic levels. These studies established the long‐term therapeutic chimeric threshold of normal white blood cells at ∼26% and determined the minimal fractionated BM/stem cell doses concomitant with mild myelosuppression for significant correction of SCD in SAD mice.

670. Combined Strategy of Minimal Myeloablation and Optimal BMT Regimen To Cure Murine β-Thalassemia

Using a thalassemic mouse model (hemi-|[beta]|thal mouse) deleted from the two adult genes in the |[beta]|-globin locus and reproducing the human |[beta]|-thalassemia intermedia, we have recently established by a competitive repopulating assay that only 19 to 24% normal bone marrow cells transplanted into lethally irradiated hosts was sufficient to significantly correct the disease. Based on this finding, we hypothesized that a minimal level or absence of myeloablation could be used for therapeutic correction of |[beta]|-thalassemia and thus, investigated this minimal level as well as the optimal cell transfer conditions to achieve such treatment. A series of bone marrow transplantations was performed on 51 partially irradiated (100 or 200 Rads) hemi-|[beta]|thal mice divided into 8 groups of 5 to 8 chimeric mice each. The hemi-|[beta]|thal mice received 20, 40, or 60 million nucleated bone marrow cells in either one dose or two equal doses given at 24h difference. Engraftment and chimerism of hemi-|[beta]|thal recipient mice were analyzed for short and long-term post- transplantation (2 months to >6 months) using the Gpi1a marker. When one cell dose was given to mice conditioned with 100 Rads, no mice (0/6) engrafted with 40 million cell whereas 42.8% of mice (3/7) engrafted with 60 million cells at 23% mean chimerism in the latter group. Of interest, mice conditioned with 200 Rads showed significantly more engraftment from 50% (4/8 mice, 20 million cells) to 85% (6/7 mice, 40 million cells) leading to |[sim]|36% and |[sim]|66% mean chimerism, respectively. Most importantly, when mice conditioned with 100 Rads received two half-doses of cells at 24 hour difference, the engraftment increased from 42.8% (3/7 mice, 2 |[times]| 20 million cells) to 100% (6/6 mice, 2 |[times]| 30 millions cells) with 40 and 48% mean chimerism respectively, while mice conditioned with 200 Rads showed 80% engraftment (4/5 mice) for 20 and 40 million cells transferred, with 63 and 84% mean chimerism respectively. Mice with 23% bone marrow chimerism showed significant improvement of most hematological parameters on complete blood count (CBC) and of all parameters with chimerism greater than 36%, consistent with our recent findings. Altogether, our results established 100 Rad irradiation and two-cell doses as the minimal therapeutic requirements for optimal engraftment and chimerism. Significantly, this is the first study to determine a transplantation strategy in minimally ablated thalassemic mice to attain 100% engraftment efficiency and a therapeutic chimerism in all mice, constituting a basis amenable to clinical applications for human thalassemia.