Journal of Blood Disorders Symptoms and Treatments

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Research Article

Microparticles from Children with Sickle Cell Anemia and the Acute Chest Syndrome Cause Activation of Human Pulmonary Endothelium

Christopher McKinney, Marguerite R Kelher, Rachelle Nuss, Alysse Cool, Alex Hoffman, Christopher C Silliman

Correspondence Address :

Christopher C Silliman
MD, PhD, Senior Independent Investigator
Research Laboratory
Bonfils Blood Center
717 Yosemite Street, Denver, CO 80230, USA
Email: Christopher.Silliman@ucdenver.edu

Received on: February 28, 2018, Accepted on: March 09, 2018, Published on: March 16, 2018

Citation: Christopher McKinney, Marguerite R Kelher, Rachelle Nuss, et al. (2018) Microparticles from Children with Sickle Cell Anemia and the Acute Chest Syndrome Cause Activation of Human Pulmonary Endothelium

Copyright: 2018 Christopher C Silliman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Children with sickle cell anemia (SCA) have increased numbers of microparticles (MPs) some of which increase with known SCA complications. Statins have pleiotropic effects including inhibition of MP formation. We hypothesize that MPs increase in children with SCA, depending upon their disease state, and cause pro-inflammatory activation of vascular endothelium which is statin-inhibitable. Blood was drawn from children with SCA at clinic, when they were well (resting) and upon hospital admission for vaso-occlusive crisis (VOC) or with the acute chest syndrome (ACS). MPs were isolated by centrifugation and classified with fluorescently-labelled antibodies and flow cytometry. Pulmonary human microvascular endothelial cells (HMVECs) were incubated with MPs and intercellular adhesion molecule-1 (ICAM-1) surface expression measured by flow cytometry with chemokine release measured by ELISA. VOC caused increased red blood cell (RBCs) plasma MPs, while ACS induced increased MPs from platelets, leukocytes, and RBCs versus resting samples.
The MPs from ACS elicited increased HMVEC ICAM-1 surface expression and release of IL-8 and growth-related oncogene- α (Gro-α). Simvastatin abrogated HMVEC activation. We conclude that VOC and ACS increase plasma MP formation, and the ACS MPs caused pro-inflammatory activation of HMVECs that were statin-inhibitable. Statin treatment may decrease ACS, and a clinical trial is warranted.

Keywords: Intercellular adhesion molecule-1, Interleukin-8, Growth-related oncogene-α, Statins


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 [6].
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 [12]. 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.

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 [16]. 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 [16].

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 [17]. 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 [17]. Interleukin-8 (IL-8) and growth-related oncogene-α (Gro-α) concentrations were measured in the stored supernatants by ELISA [17].


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).

Microparticles Induce Pro-inflammatory Activation of Pulmonary Endothelial Cells

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 [14]. 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 [24]. When used in vitro statins also decrease the adhesion of neutrophils from SCA patients to TNFα-activated endothelium acutely similar to the presented data [22]. 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 [27]. 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.
1. Haywood C Jr, Tanabe P, Naik R, Beach MC, Lanzkron S. The impact of race and disease on sickle cell patient wait times in the emergency department. Am J Emerg Med. 2013;31(4):651-656.
2. Mayer ML, Konrad TR, Dvorak CC. Hospital resource utilization among patients with sickle cell disease. J Health Care Poor Underserved. 2003;14(1):122-135.
3. Bainbridge R, Higgs DR, Maude GH, Serjeant GR. Clinical presentation of homozygous sickle cell disease. J Pediatr. 1985;106(6):881-885.
4. DeBaun MR, Strunk RC. The intersection between asthma and acute chest syndrome in children with sickle-cell anaemia. Lancet. 2016;387(10037):2545-2553.
5. Vichinsky E, Styles L. Pulmonary complications. Hematol Oncol Clin North Am. 1996; 10(6):1275-1287.
6. Charache S, Terrin ML, Moore RD, et al . Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N Engl J Med. 1995;332(20):1317-1322.
7. Mobarrez F, He S, Broijersen A, et al. Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on plateletderived micro particles in patients with peripheral arterial occlusive disease. Thromb Haemost. 2011;106(2):344-352.
8. Nomura S, Shouzu A, Omoto S, et al. Effects of losartan and simvastatin on monocyte-derived micro particles in hypertensive patients with and without type 2 diabetes mellitus. Clin Appl Thromb Hemost. 2004;10(2):133-141.
9. Nomura S. Statin and endothelial cell-derived micro particles. Thromb Haemost. 2008; 100(3):377-378.
10. Nomura S, Shouzu A, Omoto S, et al. Effects of pitavastatin on monocyte chemoattractant protein-1 in hyperlipidemic patients. Blood Coagul Fibrinolysis. 2009;20(6):440-447.
11. Nomura S, Inami N, Shouzu A, et al. The effects of pitavastatin, eicosapentaenoic acid and combined therapy on platelet-derived micro particles and adiponectin in hyperlipidemic, diabetic patients. Platelets. 2009;20(1):16-22.
12. Gerotziafas GT, Van DP, Chaari M, et al. The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived micro particles. Thromb Haemost. 2012;107(6):1044-1052.
13. Shet AS, Hoffmann TJ, Jirouskova M, et al. Morphological and functional platelet abnormalities in Berkeley sickle cell mice. Blood Cells Mol Dis. 2008;41(1):109-118.
14. Tantawy AA, Adly AA, Ismail EA, Habeeb NM, Farouk A. Circulating platelet and erythrocyte micro particles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications. Platelets. 2013;24(8):605-614.
15. van Tits LJ, van Heerde WL, Landburg PP, et al. Plasma annexin A5 and micro particle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis. Biochem Biophys Res Commun. 2009;390(1):161-164.
16. Kent MW, Kelher MR, West FB, Silliman CC. The pro-inflammatory potential of micro particles in red blood cell units. Transfus Med. 2014;24(3):176-181.
17. Wyman TH, Bjornsen AJ, Elzi DJ, et al. A two-insult in vitro model of PMNmediated pulmonary endothelial damage: requirements for adherence and chemokine release. Am J Physiol Cell Physiol. 2002;283(6):C1592-C1603.
18. Silliman CC, Curtis BR, Kopko PM, et al. Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event in vitro model. Blood. 2007;109(4):1752-1755.
19. Albelda SM, Smith CW, Ward PA. Adhesion molecules and inflammatory injury. FASEB J. 1994;8(8):504-512.
20. Ball JB, Khan SY, McLaughlin NJ, et al. A two-event in vitro model of acute chest syndrome: the role of secretory phospholipase A2 and neutrophils. Pediatr Blood Cancer. 2012;58(3):399-405.
21. Salzer WL, McCall CE. Primed stimulation of isolated perfused rabbit lung by endotoxin and platelet activating factor induces enhanced production of thromboxane and lung injury. J Clin Invest. 1990;85(4):1135-1143.
22. Canalli AA, Proenca RF, Franco-Penteado CF, et al. Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin. Haematologica. 2011;96(4):526-533.
23. Hoppe C, Kuypers F, Larkin S, et al. A pilot study of the short-term use of simvastatin in sickle cell disease: effects on markers of vascular dysfunction. Br J Haematol. 2011; 153(5):655-663.
24. Hoppe C, Jacob E, Styles L, et al. Simvastatin reduces vaso-occlusive pain in sickle cell anaemia: a pilot efficacy trial. Br J Haematol. 2017;177(4):620-629.
25. Rosch JW, Boyd AR, Hinojosa E, et al. Statins protect against fulminant pneumococcal infection and cytolysin toxicity in a mouse model of sickle cell disease. J Clin Invest. 2010;120(2):627-35.
26. Solovey A, Kollander R, Shet A, et al. Endothelial cell expression of tissue factor in sickle mice is augmented by hypoxia/reoxygenation and inhibited by lovastatin. Blood. 2004;104(3):840-846.
27. Adam SS, Hoppe C. Potential role for statins in sickle cell disease. Pediatr Blood Cancer. 2013;60(4):550-557.
Tables & Figures

Figure 1: Microparticle distribution in children with SCA as a Reflection of their Physiologic State.
Panel A: Microparticles (MPs) were isolated from cell-free supernatants from all children with SCA in different physiologic states: resting, VOC, and ACS. The MPs were incubated with fluorescent antibodies for platelets (Plts, CD41), white blood cells (WBCs, CD45), red blood cells (RBCs, CD235), and endothelial cells (CD54) and the amounts measured by flow cytometry. MPs were significantly increased, from resting, in ACS patients from Plts, WBCs, and RBCs (* p<0.05) (n=11) (A). In addition, only the MPs from WBCs from children with ACS significantly increased in comparison to the WBC MPs from children with VOC (# p<0.05) (n=11) (A).
Panel B: Isolated MPs from 3 patients who presented with all three physiologic state demonstrated that the MPs from WBCs and RBCs in children with ACS were significantly increased versus the MPs from the resting state (n=3) (*p<0.05). In addition the MPs from RBCs from the 3 children with ACS were also significantly increased in comparison to the RBC MPs in these same patients
with VOC (n=3) (# p<0.05).

Figure 2: Microparticles from children with ACS cause increased ICAM-1 surface expression on pulmonary HMVECs. As compared to the media controls and micro particles (MPs) from children in the resting state, both the positive control, endotoxin (LPS), and the MPs from children with ACS elicited significant increases in ICAM-1 surface expression, black bars (n=18) (*p<0.05). However
the MPs from children with VOC did not. When the HMVECs were preincubated with 10 μM simvastatin the increased MP-elicited ICAM-1 surface expression was significantly inhibited (n=18) (# p<0.05) whereas the LPSinduced, increased ICAM-1 surface expression was unaffected (gray bars).

Figure 3: Microparticles from children with ACS caused increased chemokine release from pulmonary HMVECs.
Panel A: MPs from children with ACS caused increased IL-8 release from pulmonary HMVECs as compared to MPs from children in resting and during VOC (black bars) (n=18) *=p<0.05) Pre-incubation of HMVECs with 10 μM simvastatin returned the IL-8 release to the resting and VOC concentrations (black bars).
Panel B: MPs from children with ACS induced increased concentrations of released Gro-α versus the MPs from children in the resting, physiologic state, black bars (n=18) (p<0.05). Although the MPs from children with VOC caused an increase in released Groα, the amount of Gro-α data was not statistically different from the amount released from HMVEC from the resting or ACS MPs (black bars). Pre-incubation of HMVECs with 10 μM simvastatin significantly inhibited the increased ACS MP-elicited Gro-α release (black bars) (n=18) (=p<0.05).

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