INTRODUCTION

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Acute and chronic pulmonary disease represent the most common cause of death in adults with sickle cell disease (SCD) (1). The acute chest syndrome (ACS), characterized by fever, cough, pleuritic chest pain, and pulmonary infiltrates, occurs in 30-40% of patients with SCD (2, 3); hypoxemia is common and respiratory failure, as well as multiorgan dysfunction, may ensue (4).

Pathophysiologic mechanisms leading to development of ACS remain unclear. Infection is commonly associated with ACS in the pediatric population but does not play a major role in adults (5, 6). Moreover, thromboembolic phenomena occur no more frequently in adults with ACS than in the general population (7). Thus, microvascular occlusion due to sickled erythrocytes and/or bone marrow fat emboli is currently felt to play a significant pathogenic role in ACS (7, 8). Since repeated episodes of ACS are a major risk for development of pulmonary hypertension, cor pulmonale, and early death (9), better understanding of the underlying mechanism is needed.

The contribution of vascular endothelium to the vasoocclusive state has been suggested by several studies demonstrating increased adherence between sickled erythrocytes and the endothelium, particularly during vasoocclusive crisis (VOC) (12). Besides alterations in adherence, instability in local vascular tone via altered metabolism of endothelial-derived vasoactive mediators could contribute to vasoocclusive events. For example, plasma levels of endothelin-1 (ET-1), the most potent endothelial-derived vasoconstrictor (15), are elevated in patients with SCD (16). In the current study we investigated the role of ET-1 in ACS and found that ET-1 production in vivo and in vitro correlated with the development and stage of ACS in two patients with SCD.

	METHODS

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Patient #1

A 31-yr-old black female with homozygous SS disease presented with cough, pleuritic chest and back pain. Physical examination revealed a temperature of 38.5° C, bibasilar rales, and tachycardia. Hematocrit was 18% with a reticulocyte count of 27; hemoglobin S level was 90%. The Pa_O2 with the patient breathing room air was 77 mm Hg with an A-a gradient of 20. Admission chest roentgenogram revealed cardiomegaly with increased interstitial markings. Electrocardiogram demonstrated sinus tachycardia and no ischemic changes. On Day 3 of hospitalization, chest roentgenogram demonstrated a new right lower lobe infiltrate; empiric antibiotics were begun. Because of worsening hypoxemia and radiographic abnormalities consistent with pulmonary edema, the patient was transferred to the medical Intensive Care Unit (MICU), and right heart catherization performed. Central venous pressure was 8 mm Hg, pulmonary artery systolic pressure 35 mm Hg, pulmonary capillary wedge pressure 8 mm Hg and cardiac output 12.0 L/min. Diagnosis of ACS was made and exchange transfusion totalling 4 units initiated with slow clinical improvement. During subsequent clinic visits, the patient reported occasional back and lower extremity discomfort but did not require hospitalization. Six months after the previous hospitalization and four days after a routine clinic visit, the patient was hospitalized with severe lower extremity, back, and chest pain.

Patient #2

A 21-yr-old black female with homozygous SS disease presented with diffuse back pain. Physical examination revealed a temperature of 38.6° C, clear lung fields and tachycardia. Hematocrit was 20.7 with a reticulocyte count of 25; hemoglobin S level was 90%. Admission chest roentgenogram was normal; SaO₂ with the patient breathing room air was 99%. On Day 3 of hospitalization, left pleuritic chest pain developed, and a chest roentgenogram revealed a left lower lobe infiltrate and bilateral pleural effusions. Because of worsening hypoxemia, she was transferred to the MICU; central venous pressure was 6 mm Hg and repeat hemoglobin S was 95%. Diagnosis of ACS was made and the patient underwent exchange transfusion totalling 6 units with marked clinical improvement. During clinic visits over the next 6 mo, the patient remained asymptomatic.

Blood Samples

Peripheral blood samples were collected in heparinized syringes during acute (before exchange transfusion) and convalescent stages of ACS and during routine clinic visits (approved by Boston City Hospital Human Studies Committee and informed written consent obtained from subjects). Samples were placed on ice and centrifuged at 2,000 × g for 15 min. Plasma centrifuged at 20,000 × g for 20 min provided platelet poor plasma. Peripheral blood collected similarly from normal volunteers served as control. Aliquots were preserved at 70° C until assays were performed.

Cell Cultures

Bovine pulmonary artery endothelial cells (BPAEC) were cultured in MEM containing 15% heat inactivated bovine calf serum (17, 18). Purity was verified by "cobblestone" appearance and labelling with fluorescent acetylated low density lipoprotein. BPAEC from passage five from several different primary cell lines were used in these experiments.

Experimental Protocol

BPAEC grown to confluence in 35 mm dishes were incubated for up to 20 h with normal or sickle red blood cells (RBCs; final concentration equivalent to a hematocrit of 20%) suspended in MEM and/or autologous plasma (1:10 dilution).

RNA Isolation and Northern Analysis

Total cellular RNA was isolated using TriReagent (MRC, Cincinnati, OH) followed by chloroform extraction, isopropanol, and ethanol precipitation (18). RNA was separated on a 1% agarose gel and transferred to a nitrocellulose membrane (Hybond, Amersham, UK). The filter was hybridized with a cDNA specific for the human preproendothelin-1 gene radiolabeled via random priming; -actin (gift from Dr. A. Fine, Boston University School of Medicine) served as an internal control. Densitometry of autoradiographs used a Molecular Dynamics Computing Densitometer (Sunnyvale, CA).

Determination of ET-1 Levels

ET-1 levels were measured in triplicate in plasma and BPAEC supernatants by radioimmunoassay (RIA) according to manufacturer's instructions (Amersham, UK).

	RESULTS

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ET-1 mRNA Levels

Sickle RBCs and/or plasma obtained during the acute stage of ACS upregulated ET-1; this effect was greatest following exposure to plasma alone (Figure 1). Therefore, in subsequent experiments, sickle plasma alone was used. BPAEC ET-1 mRNA expression was increased following exposure to plasma obtained from both patients throughout acute stages of ACS (Figure 2). In contrast, exposure to plasma from either patient obtained during a routine clinic visit 3 to 6 mo after discharge did not increase BPAEC ET-1 mRNA. Interestingly, the greatest increase in BPAEC ET-1 mRNA expression occurred after exposure to plasma obtained from Patient 1, 4 d prior to hospitalization for VOC.

View larger version (66K): [in this window] [in a new window]   Figure 1.   ET-1 mRNA expression in BPAEC exposed to sickled RBCs and/or plasma obtained during ACS. Lane 1 = control (BPAEC alone); Lane 2 = RBCs from normal volunteer; Lane 3 = RBCs and plasma from normal volunteer; Lane 4 = plasma from normal volunteer; Lane 5 = sickled RBCs from patient with ACS (MICU admission); Lane 6 = sickled RBCs and plasma from patient with ACS (MICU admission); Lane 7 = plasma from patient with ACS (MICU admission).

View larger version (46K): [in this window] [in a new window]   View larger version (65K): [in this window] [in a new window]   Figure 2.   ET-1 mRNA expression in BPAEC exposed to serial plasma samples. (A) Patient 1. Top panel = ET-1; middle panel = -actin; lower panel = densitometric analysis of ET-1 induction compared to -actin. Control (BPAEC alone); ACS = acute chest syndrome (MICU admission); ACS Day 3 = acute chest syndrome (MICU Day 3); 3 mo F/U = stable clinic visit 3 mo after discharge; 6 mo F/U = 4 d prior to hospitalization for VOC. Northern blot analyses of ET-1 mRNA were performed as outlined in the METHODS. (B) Patient 2: densitometric analysis of ET-1 induction compared to -actin. cl (BPAEC alone); Day 1 = acute chest syndrome (MICU admission); Day 2 = acute chest syndrome (MICU Day 2); Day 3 = acute chest syndrome (MICU Day 3); 6 mo F/U = stable clinic visit 6 mo after discharge. Northern blot analyses of ET-1 mRNA and densitometric analysis of ET-1 induction were performed as outlined in METHODS.

ET-1 Protein Levels in Culture Supernatants

In each case, release of ET-1 paralleled the change in ET-1 mRNA expression (example in Figure 3). ET-1 protein level was elevated following exposure to plasma obtained during the initial stage of ACS and even higher in supernatants of BPAEC exposed to plasma obtained on Day 3 of hospitalization. ET-1 protein levels in supernatants of BPAEC exposed to plasma obtained during a routine clinic visit 3 to 6 mo after discharge were similar to control levels. Interestingly, maximum ET-1 levels were found in supernatants of BPAEC exposed to plasma obtained from Patient 1, 4 d prior to hospitalization for VOC.

View larger version (22K): [in this window] [in a new window]   Figure 3.   ET-1 levels in supernatants of BPAEC after exposure to serial plasma samples. ET-1 levels were measured as outlined in the METHODS. Control (BPAEC alone); ACS = acute chest syndrome (MICU admission); ACS Day 3 = acute chest syndrome (MICU Day 3); 3 mo F/U = stable clinic visit 3 mo after discharge; 6 mo F/U = 4 d prior to hospitalization for VOC.

ET-1 Levels in Plasma

Plasma ET-1 level was markedly elevated during the initial stage of ACS and decreased by Day 3 of hospitalization (example in Figure 4). Plasma ET-1 level measured during a routine clinic visit 3 to 6 mo after discharge remained slightly elevated. Interestingly, the plasma ET-1 level had increased markedly in Patient 1, 4 d prior to hospitalization for VOC.

View larger version (16K): [in this window] [in a new window]   Figure 4.   ET-1 plasma levels during various clinical states. ET-1 levels were measured as outlined in the METHODS. Control (normal volunteer); ACS = acute chest syndrome (MICU admission); ACS Day 3 = acute chest syndrome (MICU Day 3); 3 mo F/U = stable clinic visit 3 mo after discharge; 6 mo F/U = 4 d prior to hospitalization for VOC.

	DISCUSSION

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In this report, we demonstrate that BPAEC ET-1 mRNA and protein expression in vitro following exposure to plasma obtained serially from patients with SCD and ET-1 levels in vivo correlate with stage of disease. This effect occurs in the absence of direct erythrocyte contact in vitro and suggests that a soluble plasma factor precedes and is associated with development of ACS or VOC.

Pathophysiologic events leading to development of ACS have not been determined but are likely similar to those observed in other organ systems. Until recently, study of vasoocclusive events had focused on the microvasculature; it is now appreciated that large vessel occlusion and damage are responsible for the cerebrovascular accidents and pulmonary infarctions observed in SCD (7, 19, 20). The role of the endothelium in the pathogenesis of microvascular occlusion was first suggested by ultrastructural studies demonstrating sickle RBC adherence to the endothelium (21). Subsequent static and dynamic models in multiple species have confirmed increased sickle RBC adherence, especially to postcapillary venular endothelium (12, 22). Although precise mechanisms involved in increased adherence of sickled RBCs to endothelium have not been clarified, adherence parallels severity of disease and is increased by addition of autologous plasma obtained during VOC (13, 23).

In a similar fashion, ACS likely involves perturbations of normal homeostatic functions of pulmonary vascular endothelium. In addition to adherence, interaction of sickle RBCs and/or plasma factors with EC may alter endothelial production of vasoactive mediators (24). For example, abnormalities in oscillatory flow during the steady state as well as absence of normal physiologic responses in vascular flow to autonomic stimuli are found in patients with SCD (25). Furthermore, nailbed capillary flow studies reveal that sickle RBC velocity and flow are abnormal during crisis state (26). Inability to maintain a balanced microvascular state results in trapping of sickle RBCs leading to microvascular occlusion and crisis, particularly within organ systems with microcirculatory responses sensitive to flow and hypoxia, such as the lung. Thus, a vicious cycle of sickling, tissue hypoxia, altered EC production of vasoactive mediators and obstructed microcirculatory flow could be created, leading to eventual obliteration of the vascular bed and pulmonary hypertension.

Endothelial cell molecules important for maintenance of normal vascular tone such as prostaglandins, ET-1 and nitric oxide may be altered during steady state or crisis in SCD (16, 24, 27). For example, prostaglandin metabolism is modulated by sickle RBCs in vitro and, by a small degree, in vivo (27). Elevated circulating levels of ET-1 have been found in patients with SCD during steady state and crisis periods (16). Furthermore, ET-1 gene activation has been demonstrated in human umbilical vein EC exposed to RBCs sickled in vitro to simulate VOC (30). In the current study, we used sickle RBCs and plasma from patients during acute and convalescent stages of ACS, as well as prior to VOC, and found that ET-1 mRNA expression in pulmonary artery EC and that levels of ET-1 protein in vitro, as well as in vivo, correlate with stage of disease. Thus, our study extends these phenomena to both an in vitro and in vivo investigation of acute and convalescent stages of ACS.

Although these observations, both in vitro and in vivo, only encompass two patients and do not prove causality, they suggest that upregulation of the vasoconstrictor ET-1 participates in the early phase of development of ACS and VOC and may contribute to the initial microvascular occlusive state leading to ACS. This ET-1 production occurs independent of direct erythrocyte contact, implicating the presence of a soluble plasma factor. If validated in subsequent studies, elevation in ET-1 plasma levels we observed immediately prior to development of ACS or VOC may identify patients benefitting from early treatment, potentially preventing prolonged hospitalization and early death. Further studies to characterize and clarify the role of ET-1 and to identify the soluble plasma factor responsible for its upregulation will improve our understanding of the altered endothelial state in SCD.