Journal of Thrombosis and Thrombolysis 19(2), 83–86, 2005. C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.


Endothelial Function in Patients with Sickle Cell Anemia During and After Sickle Cell Crises Arnon Blum, MD,1 Shay Yeganeh, MD,1 Aviva Peleg, MSc,2 Fina Vigder, MD,3 Konstantin Kryuger, MD,3 Ahmed Khatib, MD,1 Khalid Khazim, MD,1 Harold Dauerman, MD4 1 Department

of Internal Medicine A, 2 the Biochemical Research Laboratory, and the 3 Department of Roentgenology, Poria Medical Center, Israel; 4 The Cardiology Department, Vermont University, Burlington, VT, USA

Abstract. Background: Prior studies have demonstrated increased adherence of sickle cell erythrocytes to vascular endothelial cells. While decreased production of nitric oxide and increased production of adhesion molecules have been implicated in this pathophysiology, the relative contribution of these mechanisms during acute sickle cell crises as compared to steady state conditions have not been elucidated. Methods and results: We studied 10 consecutive young adult patients presenting with a sickle cell crisis. Endothelial function was evaluated by a noninvasive brachial artery shear stress method. Serum levels of adhesion molecules were obtained during the crisis. Both brachial artery responsiveness and serum levels of adhesion molecules were then repeated at steady state. Ten age and gender matched volunteers served as a control group. Impaired endothelial function and impaired endothelium-independent vasodilatation were observed in all sickle cell patients during both steady state and during crisis. Flow mediated dilation (FMD)% was 3.25 ± 2.76% during crisis, 4.57 ± 4.11 at steady state, compared with the control group FMD of 11.64 ± 7.69% ( p < 0.001). Flow independent dilation was 10.35 ± 11.3% during crisis, 10.03 ± 6.52% at steady state, compared with control group FID of 24.17 ± 11.87% ( p < 0.001). Levels of cell adhesion molecules and markers of inflammation were increased in sickle cell crisis patients compared with the control group: sCD40 ligand levels during the acute crisis were over twice the level of normal matched volunteers ( p = 0.02), and similarly significant increases were seen for E-selectin ( p = 0.008), ICAM-1 ( p = 0.037) and VCAM-1 levels ( p = 0.01). The levels of each of these biomarkers was not significantly increased during acute crises as compared to patients’ recovery state. Conclusions: Sickle cell anemia patients have severe systemic endothelial dysfunction as demonstrated by both brachial artery assessment and increased serum levels of adhesion molecules. These abnormalities characterize not only the sickle cell crisis but also the steady state pathophysiology of sickle cell anemia. Key Words. sickle cell, adhesion molecule, endothelial function

S ickle cell disease is characterized by recurrent, painful crises and organ damage resulting from microvascular occlusion. Endothelial dysfunction may be induced by sickle cells and secondary inflammatory stimuli. Subsequently, adherent leukocytes may interact with circulating sickle erythrocytes, leading to acute compromise of micro-vascular blood flow [1]. Several markers of endothelial dysfunction, activation and inflammation have been implicated in the development of vaso-occlusion and endothelial dysfunction [1–11]. For example, vascular cell adhesion molecules like E-selectin, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 may play a significant role in sickle cell crises as their activation is mediated through nitric oxide dependent pathways that have been demonstrated to be abnormal in patients with sickle cell disease. It is not clear whether the acute crisis period of sickle cell anemia is characterized by markedly worsened levels of endothelial dysfunction, as compared to the steady state. Such a finding might have significant implication for targeting therapies aimed at preventing sickle cell crises [3–11]. Thus, we investigated whether levels of endothelial dysfunction as well as serum markers of inflammation and endothelial activation varied between steady state and acute crises in patients with sickle cell anemia.

Methods Study design This was a prospective study performed at a single center that was approved by the hospital institutional review board. The diagnosis was based on hemoglobin electrophoresis (Hemoglobin F more Address for correspondence: Arnon Blum, MD, Department of Internal Medicine A, Poria Medical Center, Lower Galilee 15208, Israel. Tel/Fax: 972-4-6652687; E-mail: [email protected]

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than 50%). Ten consecutive patients presenting with sickle cell crises were enrolled after obtaining informed consent. Ten age- and gender-matched volunteers served as the control group. Endothelial function was studied vivo by the brachial artery method. Measurements were done during the acute sickle crisis and during the steady state condition (at least 2 months after the acute attack). Venous blood samples were obtained during acute sickle cell crises and during steady state. Serum markers of inflammation and endothelial activation/dysfunction included soluble CD40 ligand (sCD40L), E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). All blood samples were separated (by centrifugation) and were frozen at −70◦ C until studied as one batch at the end of the study. Levels of each biomarker were determined by ELISA (R and D Systems, Inc, Minneapolis, MN). All assays employed the quantitative sandwitch enzyme immunoassay technique. Monoclonal antibodies specific for soluble E-selectin, soluble CD40, ICAM-1 and VCAM-1 have been pre-coated onto microplates. Standards, samples, controls and conjugates were pipetted into the wells and soluble adhesion molecules presented were sandwitched by the immobilized antibody and a second enzymelinked monoclonal antibody specific for the adhesion molecule studied.

Determination of endothelial dysfunction The vascular studies were performed according to the technique of Celermajer and Deanfield [2]. All subjects lay supine, and an imaging study of the right brachial artery was performed using a highresolution ultrasound (12 MHz linear array transducer) following 30 minutes of rest. Baseline measurements included brachial artery diameter and flow velocity measured by pulse-doppler at approximately 70 degrees to the vessel. Endotheliumdependent vasodilatation (FMD%) was assessed by measuring the maximum increase in diameter of the brachial artery during reactive hyperemia created by an inflated cuff (50 mmHg above the systolic blood pressure for 5 min) on the forearm. After cuff deflation, flow velocity was measured for the first 15 seconds then the artery lumen was recorded continually for the next 90 seconds of hyperemia. After 15 minutes’ rest, basic measurements (diameter and flow) were taken, then a sub-lingual nitroglycerin tablet (0.4 mg) was given. The same measurements were repeated, reflecting the endotheliumindependent response (FID%). Statistical analysis Measurements are expressed as mean ± SD. The two-sided paired Student t test was used to compare changes in vascular responses and laboratory values between sickle cell patients at both phases (the acute

Table 1. Flow Mediated Dilation and Flow Independent Dilation During the Acute Sickle Cell Crisis, at Steady State, and in Comparison to Age/Gender Matched Controls Sickle cell patients

FMD%

FID%

Acute crisis Steady state Volunteers P -value

3.25 ± 2.76 4.57 ± 4.11 11.64 ± 7.69 0.0003

10.35 ± 11.3 10.03 ± 6.52 24.17 ± 11.87 0.0005

FMD%—Flow Mediated (Endothelium-Dependent) Dilatation. FID%—Flow Independent Dilatation (Nitroglycerine-induced stimulation of vascular smooth muscle cell dilation). P > 0.10 for comparisons between acute crises and steady state.

crisis and the steady state) and the volunteers in the control group, with p < 0.05 an indicator of statistical significance. The primary study comparison was FMD% in sickle cell anemia patients at crisis and steady state as compared to the healthy volunteers in the control group. The secondary study comparison was levels of biomarkers at crisis and steady state as compared to healthy volunteers.

Results Endothelial dysfunction in patients with sickle cell anemia Ten sickle cell anemia patients aged 18–34 years old (mean age of 26 ± 8 years, 6 men and 4 women) took part in the study. Brachial artery response measurements were done within the first 24 hours of admission. All patients were treated with hydroxyurea and folic acid. The patients had severely impaired endothelial function (FMD%) and impaired endothelium-independent vasodilatation (FID%) during crises as compared to health matched controls (P < 0.001 for both comparisons) (Table 1). On the other hand, there was no significant difference between the crisis and steady states for the FMD% and FID% ( p = 0.46 and p = 0.78, respectively). A clear gender difference was not seen for the sickle cell patient acute crisis measurements: FMD% of females (N = 6) was 4.46 ± 5.01% compared with males (n = 4) whose FMD% was 3.63 ± 2.51% ( p = 0.45). The FID% was 7.14 ± 8.63% for women and 11.07 ± 8.56% for men ( p = 0.56). Markers of endothelial activation and inflammation There were significant differences in levels of cell adhesion molecules among patients with sickle cell anemia in both crisis and steady state as compared with the control group (Table 2). Levels of sCD40L, Eselectin, VCAM-1 and ICAM-1 were approximately doubled among patients with sickle cell crises as compared to normal volunteers. There was a trend towards increased levels of E-selectin ( p = 0.09) and ICAM-1 ( p = 0.07) among sickle cell patients in acute

Endothelial Function in Patients with Sickle Cell Anemia During and After Sickle Cell Crises

Table 2. Markers of Endothelial Activation and Inflammation in Sickle Cell Anemia Patients During and After Acute Crisis and During Steady State Conditions Crisis

Steady

Volunteers

sCD40 1039 ± 374∗ 964 ± 350 523 ± 177 5.1 ± 3.0 1.9 ± 0.7 E-selectin 6.8 ± 2.8∗∗ ICAM-1 23.5 ± 10.4∗∗∗ 15.3 ± 8.1 13.7 ± 3.6 VCAM-1 22.7 ± 10.8# 19.18 ± 6.2 10.84 ± 2.46

P -value∗ 0.02 0.008 0.03 0.01

For comparison of Acute Crisis versus Steady State, p = 0.9 for sCD40L, 0.09 for E selectin, 0.07 for ICAM-1 and 0.54 for VCAM-1. *P -value is compared between the Acute Crisis results and the Volunteers’ results.

crises versus steady state, while the levels of sCD40L and VCAM-1 were remarkably similar between the two sickle cell states.

Discussion Patients with sickle cell anemia have severe endothelial dysfunction and vascular smooth muscle cell dysfunction. Abnormalities of nitric oxide dependent vasodilation have been previously described among patients with sickle cell anemia [3–10]; our results expand upon these prior findings by demonstrating the similar levels of dysfunction occurring during both acute sickle cell crises and during steady state conditions. Furthermore, our findings confirm the relationship between the brachial artery assessed systemic vascular dysfunction and heightened levels of biomarkers known to confer increased risk of endothelial dysfunction, inflammation and endothelial cell activation. Interactions between activated endothelium and sickle cell erythrocytes contribute to the vascular complications of sickle cell disease. Endotheliumderived nitric oxide plays a major role in the regulation of vasomotor tone in response to wall shear stress variations and pharmacologic stimuli [3]. Previous studies have shown FMD impairment and abnormal vasoconstrictive responses in patients with sickle cell anemia [4]. Furthermore, adhesion of sickle erythrocytes to vascular endothelium plays a central role in sickle cell complications. Cytokines and adhesion molecules are critically involved in the regulation of the adhesive processes. It has been previously demonstrated that asymptomatic sickle cell patients have a high serum level of sVCAM-1. In patients having vaso-occlusive crisis, VCAM-1 levels increased more and seemed to correlate with crisis evolution [11]. Our results extend these prior observations by comparing multiple biomarkers as well as brachial artery reactivity during both crises and steady state conditions. In our study, we have demonstrated high levels of both cell adhesion molecules and inflammatory biomarkers (soluble CD40 ligand) among patients in sickle cell crisis as well as at steady state.

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These findings are consistent with prior in vitro results. It has been demonstrated that sickle cell adherence to the endothelium occurs only following endothelial activation with TNF-alpha or addition of thrombospondin [5]. This phenomenon is nitric oxide dependent since it has been shown that sickle monocytes triggered endothelial nuclear factor kappa B (NFkB) nuclear translocation [6]. Furthermore, the pathophysiologic role of these factors is supported by studies in which antibodies to tumor necrosis factor alpha and interleukin1-beta blocked activation of the endothelium by monocytes. Unlike prior studies of VCAM-1 in sickle cell patients [11], our findings do not suggest a more intense endothelial cell activation during the acute crisis versus the steady state (as measured by E-selectin and ICAM-1 levels).

Study limitations The study group was small and the follow-up time was short; a larger study would be needed to definitively conclude the importance of this change and the relative contribution of each adhesion molecule to the acute changes occurring during vaso-occlusive crises. Conclusions Abnormalities in endothelial performance and heightened markers of cellular adhesion are present among patients with sickle cell disease.

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