Murine models of sickle cell disease and beta-thalassemia demonstrate pulmonary hypertension with distinctive features

Abstract

Sickle cell anemia and β-thalassemia intermedia are very different genetically determined hemoglobinopathies predisposing to pulmonary hypertension. The etiologies responsible for the associated development of pulmonary hypertension in both diseases are multi-factorial with extensive mechanistic contributors described. Both sickle cell anemia and β-thalassemia intermedia present with intra and extravascular hemolysis. And because sickle cell anemia and β-thalassemia intermedia share features of extravascular hemolysis, macrophage iron excess and anemia we sought to characterize the common features of the pulmonary hypertension phenotype, cardiac mechanics, and function as well as lung and right ventricular metabolism. Within the concept of iron, we have defined a unique pulmonary vascular iron accumulation in lungs of sickle cell anemia pulmonary hypertension patients at autopsy. This observation is unlike findings in idiopathic or other forms of pulmonary arterial hypertension. In this study, we hypothesized that a common pathophysiology would characterize the pulmonary hypertension phenotype in sickle cell anemia and β-thalassemia intermedia murine models. However, unlike sickle cell anemia, β-thalassemia is also a disease of dyserythropoiesis, with increased iron absorption and cellular iron extrusion. This process is mediated by high erythroferrone and low hepcidin levels as well as dysregulated iron transport due transferrin saturation, so there may be differences as well. Herein we describe common and divergent features of pulmonary hypertension in aged Berk-ss (sickle cell anemia) and Hbb^th/3+ (intermediate β-thalassemia) mice and suggest translational utility as proof-of-concept models to study pulmonary hypertension therapeutics specific to genetic anemias.

Keywords: heart; hemaglobinopathies; lung; metabolomics; pulmonary vascular disease.

Fig. 1.

Berk-ss and Hbb^th/3+ mice develop pulmonary hypertension with remodeling: (a) Right ventricular systolic pressures. (b) Pulmonary vascular remodeling. (c) right ventricular weight. (d) Right ventricular to body weight ratio. (e) Left ventricle plus septum weight. (f) Fulton index (RV/LV+S). (g) Iron loaded cells accumulate in the lungs of Berk-ss and Hbb^th3/+ mice. *p < 0.01 vs. wild type; **p < 0.05 vs. wild type t-test; ***p = 0.013 vs. Hbb^th/+. L: vessel lumen; black arrows showing iron-loaded cells. (h) Mild RV interstitial fibrosis following Masson’s trichrome staining.

Fig. 2.

Berk-ss and Hbb^th/3+ mice develop right ventricular mechanical dysfunction: (a) Representative tracings and corresponding schematics of pressure volume (PV) loop relationships during an occlusion for wild type, Hbb^th3/+, and Berk-ss mice. (b–j) Right ventricular functional analysis for stiffness, contractility, afterload, stroke volume, heart rate, cardiac output, right ventricle to pulmonary vascular coupling ratio, preload volume, and ejection fraction. *p < 0.01 vs. wild type; **p < 0.05 vs. wild type t-test; ^†p < 0.01 vs. Hbb^th/+.

Fig. 3.

Hbb^th/3+ mice develop an intermediate anemic phenotype: (a) body weight, (b) spleen weight, and (c) hematocrit. *p < 0.007 vs. wild type; **p < 0.013 vs. Hbb^th/+.

Fig. 4.

Berk-ss and Hbb^th/3+ mice demonstrate unique lung and right ventricular metabolism across the 25 most significantly changed metabolites: Multivariate analyses of metabolomics data of lungs and right ventricle (RV) of wild type (WT – green), sickle cell disease (SCA – red) and beta-thalassemia mice (blue). Analyses included principal component analysis (PCA) in (a) and (b), and hierarchical clustering analyses of the top significant 25 metabolites by ANOVA (c and d).

Fig. 5.

Lung tissue metabolites that detail primary differences between Berk-ss, Hbb^th/3+ and their shared wild type: An overview of arginine metabolism (a) in lungs from wild type (green), sickle cell disease (red), and beta-thalassemia (blue) mice, one of the most affected pathways across the three groups, along with glutaminolysis/glutathione metabolism, methionine metabolism, Krebs cycle, and related carboxylic acids (e.g., 2-hydroxyglutarate – (b)). Asterisks indicate significance by ANOVA (Kruskal-Wallis with Dunn post hoc test; *p < 0.05; **p < 0.01).

Fig. 6.

Right ventricle tissue metabolites that detail primary differences between Berk-ss, Hbb^th/3+ and their shared wild type: An overview of arginine metabolism (a) in the right ventricle (RV) from wild type (green), sickle cell disease (red), and beta-thalassemia (blue) mice, one of the most affected pathways across the three groups, along with the pentose phosphate pathway, glutaminolysis/glutathione metabolism, methionine metabolism, Krebs cycle, and related carboxylic acids (e.g., 2-hydroxyglutarate - b). Asterisks indicate significance by ANOVA (Kruskal-Wallis with Dunn post hoc test; *p < 0.05; **p < 0.01; ***p < 0.001).