Sickle cell anemia (SCA) affects 1/375 African American children and accounts for 200,000 emergency department visits per year [1,2] Acute, painful vaso-occlusive crises (VOC) are the most common complication, and the acute chest syndrome (ACS), an inherent type of acute lung injury, is related to early mortality [3-5]. Hydroxyurea has decreased the amounts of these complications by decreasing RBC sickling by increasing the amounts of fetal hemoglobin per red blood cell (RBC), which decreases the propensity of these cells to sickle and induce VOC and ACS .
Microparticles (MPs) are released from cells and have been associated with a number of disease states [7-11]. Children with SCA have increased numbers of MPs as compared to age-matched controls and elevated MP concentrations have been linked to the hypercoagulable state in SCD which may explain some of the known complications . In addition, the numbers of MPs are known to increase during acute VOC as well as ACS and other cardiopulmonary complications of SCA [13-15]. Because ACS is a form of acute lung injury (ALI) and the neutrophil has been implicated in its pathogenesis, we hypothesize that MPs increase in children with SCA depending upon their disease state and cause pro-inflammatory activation of vascular endothelium which is inhibited by statin pretreatment.
Microparticles Induce Pro-inflammatory Activation of Pulmonary Endothelial Cells
Materials and Methods
All materials were purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Buffers were made from sterile water or 0.9% sterile saline for human injection (Baxter) and sterilely filtered. Human microvascular endothelial cells (HMVECs) and cell growth media were purchased from Lonza (Allendale, NJ). Antibodies for flow cytometry were purchased from BD Bioscience (San Jose, CA). ELISAs were purchased from R&D Systems (Minneapolis, MN).
Blood Collection and Micro Particle Isolation
Whole blood was collected from children (2-18 years of age) with sickle cell anemia (SCA: hemoglobin SS, SC, or S0-thalassemia) under a protocol approved by the Colorado Multiple Institute Review Board (COMIRB) at the University of Colorado Denver, Aurora, CO. Blood was collected during steady state, at their scheduled clinic visit when they are relatively well (resting), during painful vaso-occlusive crisis (VOC), or acute chest syndrome (ACS), both upon admission to the hospital. Blood was centrifuged at 5000g for 7 min to remove cells, and the supernatant removed and centrifuged at 12,500g for 6 min to remove acellular debris and contaminating platelets . Microparticles (MPs) were then isolated by centrifugation of the cell-free supernatant at 17,500g for 60 minutes at 4oC, aliquotted, and stored at -80oC. The MPs were suspended in 1.25% human serum albumin (HSA) to the starting volume of plasma. For quantification of subtypes the MPs were incubated with fluorescently antibodies to CD41 (platelets), CD45 (white blood cells), CD235 (red blood cells), or CD54 (endothelial cells) and MP subtypes were measured with a BD FACS CantoII flow cytometer, as described .
Pro-inflammatory Activation of HMVECs
Human microvascular endothelial cells (HMVECs) were grown to 80-90% confluence in EGM-2 media with growth factors, as previously described . HMVECs were incubated for 6 hours with and then the supernatant was removed and stored at -80oC. The adherent HMVECs were removed with trypsin, incubated with an antibody to ICAM-1 (CD54), and ICAM surface expression was measured via flow cytometry . Interleukin-8 (IL-8) and growth-related oncogene-α (Gro-α) concentrations were measured in the stored supernatants by ELISA .
The data are expressed as the mean +/- the standard error of the mean. Statistical differences were determined by a paired or independent analysis of variance followed by a Bonferroni or Neuman Keuls post hoc test for multiple comparisons based upon the equality of variance.
Increase in Microparticles in Sickle Cell Disease (SCD) Blood was collected from patients at their clinic visit when they are in relatively good health (resting, n=30), or when they were admitted for a painful vaso-occlusive crisis (VOC) (n=18) or acute chest syndrome (ACS) (n=18) with three children developing both VOC and ACS. In comparison to the resting samples there were significant increases in platelet MPs (CD41+, p<0.05), leukocyte MPs (CD45+, p<0.05) and RBC MPs (CD235+, p<0.05) in the plasma from ACS patients; whereas the patient's with VOC had a significant increase in RBC MPS alone (p<0.05) (Figure 1A). In addition, there was also a significant increase in leukocyte MPs on the plasma from children with ACS versus the plasma MPs from children with VOC (Figure 1A). The MPs from endothelial cells remained unchanged irrespective of child's disease state. Samples were collected from the 3 patients who developed both VOC and ACS, and there were significant increases in MPs during ACS as compared to the resting state and during painful VOC in both the leukocyte and RBC MPs (Figure 1B).
Human microvascular endothelial cells (HMVECs) were incubated for 6 hours with media alone, lipopolysaccharide (LPS) (positive control), or MPs from children: resting, VOC, or ACS with the addition of 10 μM simvastatin or a vehicle control and ICAM-1 expression measured via flow cytometry. As compared to the media control, LPS induced significantly increased the surface expression of ICAM-1, which was unaffected by simvastatin (p<0.05) (Figure 2). Furthermore the plasma MPs from children with ACS also increased ICAM-1 surface expression, vs. the media controls and MPs from children with SCA in the resting state, which was abrogated by simvastatin (p<0.05) (Figure 2). Furthermore, the MPs from children with ACS elicited significantly increased the release of both Gro-α and IL-8 into the supernatant as compared to the MPs from children at their resting state (p<0.05) (Figure 3). Pre-treatment with simvastatin also inhibited the increased release of IL-8 and Gro-α to the levels released by MPs from children in the resting state, respectively (Figure 3).
The presented data demonstrated that compared to children with SCA when they are relatively healthy (resting) that the acute onset of VOC increased the MPs from RBCs and the onset of ACS increased platelet MPs, leukocyte MPs and RBC MPs, while the endothelial cell MPs were unaffected. The MPs from children with ACS also caused pro-inflammatory activation of primary HMVECs, including both the increase in ICAM-1 surface expression and the release of two chemokines Gro-α and IL-8. Both the increased surface expression of ICAM-1 and the release of IL-8 and Gro-α were inhibited by pre treatment of HMVECs with 10 μ M simvastatin. This pro-inflammatory activation of pulmonary HMVECs by the MP-rich fraction of plasma from children with SCA at the time ACS was identified, e.g. increased ICAM-1 surface expression and release of IL-8 and Groα, results in firm adherence of leukocytes, especially neutrophils, to activated endothelial cells [17,18]. Such activation of Human pulmonary endothelial cells may be important of ACS, a type of ALI unique to patents with SCA and such activation of the pulmonary endothelium is a required event in the two event model of ALI/the acute respiratory distress syndrome (ARDS) [17-21].
The increase in MP formation in patients with ACS has been reported before in children and adolescents with SCA and was limited to platelet and RBC MPs and did not measure the MPs from leukocytes, which were increased in children with ACS, or endothelium, which were unaffected by either VOC or ACS . The statins are known to inhibit MP formation, although most of
these studies were completed in patients or animal models using chronic statin exposure rather than acute [7,8,10,11,22-26]. Statins have been used in patients with sickle cell anemia and have demonstrated efficacy in reducing VOC episodes in a pilot study . When used in vitro statins also decrease the adhesion of neutrophils from SCA patients to TNFα-activated endothelium acutely similar to the presented data . In the sickle cell transgenic mouse statins are protective against pneumococcal infection and cytotoxicity and endothelial cell expression of tissue factor [25,26].
In conclusion, two complications of SCA, acute VOC and ACS, increase MP formation in children. The MPs alone cause pro-inflammatory activation of pulmonary HMVECs and may be involved in the pathogenesis of ACS. Statins are inexpensive medications with pleiotropic effects and inhibited the observed MP-induced HMVEC activation . A clinical trial needs to be completed to assess the ability of statins to decrease two wellknown complications of SCA and improve the health of these patients.