Loss of alpha globin genes is associated with improved microvascular function in patients with sickle cell anemia
Authors: Marc Romana, Karen Reminy, Berenike Moeckesch, Keyne Charlot, Marie‐Dominique Hardy‐Dessources, Lydia Doumdo, Benoit Tressieres, Maryse Etienne‐Julan, Nathalie Lemonne, Christopher Denton, Thomas Coates, Marie Petras, Sophie Antoine‐Jonville, Philippe Connes
Journal: American Journal of Hematology (2021)

Sickle cell anemia (SCA) affects millions of people worldwide. This genetic disease is caused by a single mutation in the β-globin gene, leading to the production of the abnormal hemoglobin S (HbS), which polymerizes under deoxygenation. The resulting fragility and loss of deformability of red blood cells (RBCs) lead to chronic anemia and vaso-occlusive complications. However, this basic pathophysiology description does not fully explain the wide heterogeneity in clinical expression of the disease. While the role played by the vascular system in the physiopathology of SCA has long been neglected, its abnormal functioning is now well documented, with alterations identified at different levels.1 The progressive chronic vasculopathy results in the development of several debilitating complications in SCA, such as stroke, cerebral vasculopathy, leg ulcers or pulmonary hypertension.1 The co-inheritance of α-thalassemia trait and SCA has been found to improve hematological indices and decrease the risk of some complications. For instance, the loss of one or two α-globin genes, called α-thalassemia trait, is associated with a lower risk of cerebral vasculopathy,2 acute chest syndrome and leg ulcers,3 with a risk reduction ranging from 34 to 71%. Pivotal in-vitro studies demonstrated that the α-globin genes and α-globin protein are expressed in the endothelial wall of arterioles and resistance arteries, where the protein regulates blood vessel tone through an interaction with endothelial nitric oxide synthase (eNOS) and scavenging of nitric oxide (NO) by heme-iron.4, 5 Straub et al.6 showed that disruption of the interaction between α-globin and eNOS increased endothelial NO activity and reduced hypertension in mice. Indeed, one could hypothesize based on these findings that the vascular function in SCA patients with one or two deleted α-globin genes would be improved as compared with SCA patients carrying four functional α-globin genes. Thirty-seven SCA (all HbSS) patients (26 males/11 females; 24.1 ± 14.4 years [range: 12–58]; body mass index (BMI): 19.0 ± 3.3 kg/m2) participated in this study after written informed consent. All patients were at steady state at the time of the study (i.e., no blood transfusion within the last 3 months and absence of acute episodes of infection, vaso-occlusive crisis or acute chest syndrome for at least 2 months before inclusion in the study). As hydroxyurea (HU) is an NO donor, which may improve vascular function, patients on HU therapy were excluded from the analysis. Multiplex gap-polymerase chain reaction was used to detect the five common alpha-thalassemia deletions (−3.7 kb, −4.2 kb, −20.5 kb, SEA, MED). Nineteen SCA patients had α-trait, 15 with co-inheritance of one 3.7 kb α-globin gene deletion and four with two 3.7 kb α-globin gene deletions. The study received ethical approval (registration numbers: SAPOTILLE/HYPOSNAD project, 2009-A00211-56/2013; VASCUDREPA project 2015/108 – 2015-1011665-44). Blood was collected in EDTA tubes for the determination of several hematological/biochemical parameters and RBC deformability (Table 1). The RBC deformability was measured at 37°C and 3 Pa by laser diffraction analysis (ektacytometry), using the Laser-assisted Optical Rotational Cell Analyzer (LORCA, RR Mechatronics, Hoorn, The Netherlands). Skin blood flow was non-invasively measured with a laser Doppler flowmeter (Periflux 5000 LDPM, Perimed, Järfälla, Sweden) on the left middle fingertip distal pad (Probe 457, Perimed, Järfälla, Sweden).7 After a 10-min baseline recording, peripheral microcirculatory cutaneous blood flow and microvascular reactivity to local heating (42°C) were evaluated for 35 min. Systolic and diastolic arterial pressures were measured and the cutaneous vascular conductance (CVC) was expressed in mV/mmHg.7 The initial peak observed in the first 1–2 min of heating exposure was followed by a plateau reached 20–30 min later. The initial peak is considered as an axon reflex caused by the activation of afferent C-fibers during nociceptive stimulation.7 The late plateau is mainly related to NO-dependent vasodilation.7 Peak and plateau responses were expressed as a percentage of the baseline values. Biological and vascular function parameters were compared between SCA patients with α-trait and those without α-trait using unpaired Student t test. Multivariate linear regression analyses were performed to identify factors independently associated with microvascular function. Significance level was defined as p < .05 (SPSS, v. 20, IBM SPSS Statistics, Chicago, IL). The SCA patients with α-trait exhibited similar blood pressure, lower MCV and higher Hb and Hct than those without (Table 1). The higher Hb is assumed to be a consequence of lower hemolysis in the α-trait group, as reflected by the observed trend for lower LDH level (p = .08). The greater RBC deformability detected in SCA patients with α-trait makes the RBCs more resistant to mechanical stress when passing through small capillaries, hence decreasing the risk of disruption. Comparison between the two groups after exclusion of the four patients with two 3.7-kb α-globin gene deletions showed no difference in MCV, but RBC deformability and hemoglobin concentration were still increased in the α-trait group. Analyses of vascular function showed that basal microvascular blood flow did not differ between the two groups (Table 1). The initial blood flow peak after the initiation of local heating was not different between the two groups. In contrast, the later plateau, mainly related to NO-dependent vasodilation,7 was higher in patients with α-trait compared to those without. Interestingly, despite the limited sample size, SCA patients missing two α-globin genes had a larger plateau (+201 ± 101%) than those missing only one α-globin gene (+107 ± 48%; p < .01) or those without α-trait (+63 ± 60%; p < .001). The plateau response was still greater in patients with one α-globin gene deleted than in those without α-trait (p < .05). A multivariate model including α-trait status, age, BMI, Hb, RBC deformability and LDH showed that both α-trait status (p = .03) and BMI (p = .004) were the strongest independent predictors of the NO-dependent plateau response (R2 = 0.60; p < .01) with co-inheritance of α-thalassemia trait increasing microvascular reactivity while low BMI did the opposite. Age (p = .55), hemoglobin level (p = .21), RBC deformability (p = .99) and LDH (p = .35) were not significant. These findings support an independent role of both BMI and the α-globin protein on the regulation of NO-related vascular function in the microcirculation in SCA. Co-inheritance of alpha-thalassemia trait with SCA has been reported to be very frequent in various countries. Accumulating evidence over the past decades showed that while α-thalassemia trait would increase the risk for repeated vaso-occlusive crises and osteonecrosis in SCA, it would also decrease the risk of developing several acute and chronic complications such as cerebral vasculopathy2 acute chest syndrome and leg ulcers.3 The reasons proposed for such a protective effect would be the lower degree of hemolysis and anemia, as well as the greater RBC deformability found in SCA patients with alpha-thalassemia trait. Recently, motivated by in vitro data on α-globin in the myoendothelial junction,5, 6 Denton and colleagues showed in non-anemic, non-SCA subjects that flow-mediated vasodilation, a well-known measure of NO-mediated vascular function, was significantly better in α-trait subjects than in subjects without α-trait.8 Thus, the mechanism proposed in vitro whereby α-globin expressed in the endothelial cells would bind, react with and oxidize NO to form nitrate, thus reducing NO levels and the resultant vasodilatory effects of NO, seems to be operative not only in non-SCA subjects but also in SCA patients. Our results suggest that the protective effect of α-trait from the development of several chronic complications in SCA could, in addition to its known effects on the RBC, be due to the effect of α-globin on the vascular system through its ability to regulate the NO diffusion from the endothelium to the smooth muscle cells of the vascular wall. Moreover, high BMI, such as in metabolic diseases, is known to be associated with poor vascular function. But it appears that low BMI is also a risk factor for impaired endothelium-dependent vasodilation in the general population.9 Our results also showed an independent association between low BMI and decreased microcirculation reactivity in SCA, which may justify the need of providing dietary guidance for adequate nutritional intake in this population. Surprisingly, a previous study10 with a larger cohort of SCA patients than our work did not find any effect of α-thalassemia trait on arterial blood pressure and arterial stiffness. The present study also failed to detect any difference in arterial blood pressure between the two SCA populations. These findings could suggest no impact of the α-thalassemia trait on the vascular function, which contrasts with the results obtained with the cutaneous laser doppler flowmetry experiments. It is noteworthy that there are many studies showing a dissociation between macrocirculatory and microcirculatory function in various diseases, with microcirculatory dysfunction usually preceding macrocirculatory dysfunction. Moreover, measurements of arterial blood pressures and arterial stiffness are obtained in static condition and do not use a stimulus (here, local heating) to force the vascular system to adapt, which is quite different from the experimental conditions used in the present study. In conclusion, our results support a role of α-thalassemia trait in the modulation of microvascular reactivity in SCA, that could partly explain the protective effects of α-thalassemia trait against the development of several complications involving the vascular system in this disease. We thank Dr. Martine Torres for her help in reviewing the manuscript. The authors declare no conflict of interest. Marc Romana, Berenike Moeckesch, Keyne Charlot, Marie-Dominique Hardy-Dessources, Sophie Antoine-Jonville and Philippe Connes designed research; Marc Romana, Karen Reminy, Berenike Moeckesch, Keyne Charlot, Lydia Doumdo, Maryse Etienne-Julan, Nathalie Lemonne, Marie Petras and Sophie Antoine-Jonville performed research; Marc Romana, Sophie Antoine-Jonville and Philippe Connes analyzed data; Marc Romana, Sophie Antoine-Jonville, Christopher Denton, Thomas Coates and Philippe Connes wrote the paper; all authors read and approved the final version of the paper. The data that support the findings of this study are available from the corresponding author upon reasonable request. The data that support the findings of this study are available from the corresponding author upon reasonable request.