Circulating Endothelial Cells: A Potential Parameter of Organ Damage in Sickle Cell Anemia?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4798-4798
Author(s):  
Michiel Strijbos ◽  
Precious Landburg ◽  
Erfan Nur ◽  
Bart J. Biemond ◽  
Frank Leebeek ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Disease Severity ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Endothelial Activation ◽  
Organ Damage ◽  
Circulating Endothelial Cells ◽  
Laboratory Markers ◽  
Leukocyte Counts ◽  
Painful Crisis

Abstract Traditional laboratory markers of disease severity (Hb concentrations, HbF%, LDH concentrations and leukocyte counts) are not powerful enough to identify individuals with sickle cell disease (SCD) at high risk of complications. In several disease states, the number of circulating endothelial cells (CEC) is indicative of the extent of vascular disease and several small studies employing cumbersome techniques have shown increased CEC to occur in SCD as well. In this pilot study we determined if, by employing the CellSearch system, elevated CEC can be detected in SCD and whether the CEC count is related to the presence of organ damage, painful crisis frequency, traditional laboratory markers of disease severity as well as markers of endothelial activation. Peripheral blood was collected from 15 sickle cell patients and 15 healthy sex, age and race matched HbAA controls. Cells positive for CD146 (present on endothelial cells) are immuno-magnetically isolated and stained with nuclear DAPI and anti-CD105 (present on endothelial cells) and anti-CD45 (pan-leukocyte marker). Within the CD146 enriched cell suspension, cells meeting the DAPI+/CD146+/CD105+/CD45− criteria after image cytometry are designated CEC. CEC counts were significantly higher in sickle cell patients (12 cells/mL, IQR 8–29) as compared to controls (4 cells/mL, 3–10) (p=0.007). CEC counts were significantly higher in patients with pulmonary hypertension (PHT) (p=0.02), and increased with the presence of number of affected organs as well (0–4 involved organs, p=0.03), with the number of affected organs being the most important determinant of increased CEC numbers in SCD (R2=0.72, P=0.001). No statistically significant correlation was detected between CEC counts and markers of endothelial activation (serum sVCAM-1 and plasma VWF:Ag levels). CEC counts did not differ between patients on hydroxyurea and those who did not use hydroxyurea. No statistically significant correlations were detected between CEC counts and painful crisis frequency, Hb, HbF%, leukocyte counts, LDH concentrations. In conclusion, elevated CEC’s can be demonstrated in the clinically asymptomatic state in patients with sickle cell anemia with a validated and automated technique and, for the first time, a relation of CEC counts to SCD related organ damage is demonstrated. Furthermore, the extent of EC activation (as assessed by sVCAM-1 and VWF:Ag levels) is not necessarily related to the extent of EC damage (as assessed by CEC counts).

Circulating endothelial cells: A potential parameter of organ damage in sickle cell anemia?

2009 ◽  
Vol 43 (1) ◽  
pp. 63-67 ◽  
Author(s):  
Michiel H. Strijbos ◽  
Precious P. Landburg ◽  
Erfan Nur ◽  
Tom Teerlink ◽  
Frank W.G. Leebeek ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Organ Damage ◽  
Potential Parameter ◽  
Circulating Endothelial Cells

Increased Platelet-Derived CD40L May Modulate Endothelial Cell ICAM-1 Expression and Interact With Leukocytes In Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 974-974
Author(s):  
Vanessa Tonin Garrido ◽  
Renata Proença-Ferreira ◽  
Venina M. Dominical ◽  
Marcos André Cavalcanti Bezerra ◽  
Aderson S. Araujo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Steady State ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Repeated Measures ◽  
Conflicts Of Interest ◽  
Endothelial Activation ◽  
Surface Expression ◽  
Platelet Surface

Abstract Background Vaso-occlusive events are a major cause of morbidity in sickle cell anemia (SCA) and attributable to the abnormal adhesion of red cells and leukocytes to the endothelium. Platelets may contribute to the chronic inflammation and endothelial activation that initiates the vaso-occlusive process. We hypothesized that platelet-associated CD40 ligand (CD40L) may contribute to platelet-mediated inflammatory responses in SCA. Aims This study evaluated the platelet (PLT) release of CD40L, the expression of its receptor (CD40) on platelets, neutrophils, lymphocytes and monocytes of control individuals (CON) and SCA patients, and also the ability of platelet-derived CD40L to activate endothelial cells. Methods IL-8, soluble ICAM-1, VCAM-1 and CD40L were determined in PLT-free plasma or the supernatant of stimulated (ADP or Collagen) and unstimulated PLTs (2•10⁸/mL in Kreb’s buffer), from CON individuals and steady-state SCA patients, by ELISA. Flow cytometry was used to analyze CD40 expression on platelets, neutrophils, lymphocytes and monocytes from the peripheral blood of the study’s subjects. Human umbilical vein endothelial cells (HUVECs) were cultured (1x106cells/well; 37°C, 5% CO2) together with PLTs (3x108PLTs/well) from CON individuals or steady-state SCA patients for 24h, 37°C, 5%CO2, in the presence, or not, of blocking antibodies against CD40L. After incubation, PLTs were removed and HUVECs analyzed by flow cytometry for CD54 (ICAM-1) surface expression. Results SCA individuals presented elevated levels of plasma CD40L (724.4± 55.7 pg/ml; n=90) compared to CON (241.5±34.6 pg/ml; n=41; P<0.0001) and these levels correlated with PLT counts (rs=0.255; P=0.015). No correlation was found between plasma CD40L and plasma IL-8, ICAM-1 or VCAM-1. PLT release of CD40L (90 min, 37°C, 5%CO2) was evaluated; PLTs of SCA patients released higher quantities of CD40L (8347±1464 pg/108 PLTs; n=10) than PLTs of CON individuals (3652±568 pg/108 PLTs; n=5; P=0.019). CD40L release from SCA PLTs was augmented by incubation with collagen (P<0.001), but not ADP. Expression of the CD40 receptor on the platelet surface was elevated in the SCA group (52.4±2.7% positive cells; n=23), compared to the CON group (36.8±3.7% positive cells; n=9; P=0.005). The surface expression of CD40 was also elevated on neutrophils (SCA, 10.4±1.5% positive cells, n=14; CON, 5.5±1.1% positive cells, n=13; P=0.03), lymphocytes (SCA, 8.3±0.8% positive cells, n=16; CON, 3.6±0.4% positive cells, n=14; P<0.001) and monocytes (SCA 69.6±5.9% positive cells, n=16; CON, 49.9±5.8% positive cells, n=14; P=0.03) of SCA patients, compared to controls. ICAM-1 expression on the surface of HUVECs (Basal expression 32.8±1.8%, n=11) was significantly increased following incubation with SCA PLTs (54.0±4.8%, n=11, p<0.0001) and slightly augmented after incubation with CON PLTs (40.8±3.1%, n=11, p<0.05; Repeated measures ANOVA). Interestingly, when HUVECs and SCA PLTs were incubated with a blocking antibody against CD40L, the increase in ICAM-1 expression was significantly reversed on HUVECs (HUVECs, 28.1±0.2%, n=6; HUVECs+SCA PLTs, 42.0±3.3%, n=6; HUVECs+SCA PLTs+anti-CD40L 28.9±1.5%, n=6; P<0.01). Conclusions Plasma levels and platelet release of CD40L were found to be significantly elevated in SCA, in association with increased expressions of the CD40 receptor on SCA PLTs, neutrophils, lymphocytes and monocytes, possibly indicating a CD40L-mediated crosstalk between platelets and leukocytes in SCA. Platelets from SCA patients can induce adhesion molecule expression on the surface of endothelial cells in vitro, and this up-regulation may be modulated by platelet-derived CD40L. Results suggest that the CD40/CD40L pathway may be altered in SCA and that platelets may participate in this up-regulation. Given the potent inflammatory effect of this cytokine, a role for platelets and this cytokine in endothelial activation, inflammation and consequent vaso-occlusion, is likely. Disclosures: No relevant conflicts of interest to declare.

Sickle Cell Anemia as a Possible State of Enhanced Anti-Apoptotic Tone: Survival Effect of Vascular Endothelial Growth Factor on Circulating and Unanchored Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3824-3830 ◽  
Author(s):  
Anna Solovey ◽  
Lizhen Gui ◽  
Sundaram Ramakrishnan ◽  
Martin H. Steinberg ◽  
Robert P. Hebbel
Keyword(s):  
Extracellular Matrix ◽  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Circulating Endothelial Cells ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor ◽  
Survival Signals

The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% ± 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% ± 15% (P < .001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P = .001) higher in plasma of sickle donors (120.1 ± 81.4 pg/mL) than that of normal donors (37.6 ± 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r = .612,P = .001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of vβ3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.

Sickle Cell Anemia as a Possible State of Enhanced Anti-Apoptotic Tone: Survival Effect of Vascular Endothelial Growth Factor on Circulating and Unanchored Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3824-3830 ◽  
Author(s):  
Anna Solovey ◽  
Lizhen Gui ◽  
Sundaram Ramakrishnan ◽  
Martin H. Steinberg ◽  
Robert P. Hebbel
Keyword(s):  
Extracellular Matrix ◽  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Circulating Endothelial Cells ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor ◽  
Survival Signals

Abstract The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% ± 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% ± 15% (P &lt; .001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P = .001) higher in plasma of sickle donors (120.1 ± 81.4 pg/mL) than that of normal donors (37.6 ± 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r = .612,P = .001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of vβ3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.

Elevated Circulating Stromal Derived Factor-1 Levels in Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1438-1438
Author(s):  
Precious Landburg ◽  
Erfan Nur ◽  
Naomi Maria ◽  
Bart J. Biemond ◽  
Dees P.M. Brandjes ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Organ Damage ◽  
Cell Disease ◽  
Steady State Levels ◽  
Leukocyte Counts ◽  
Painful Crisis

Abstract Inflammation and angiogenesis are of importance in the pathophysiology of sickle cell disease (SCD). Recently, the chemokine Stromal Derived Factor-1 (SDF-1) has been shown to be a key mediator of angiogenesis and inflammation. In this study we determined serum SDF-1 levels in consecutive adult sickle cell patients during the clinically asymptomatic state as well as during painful crisis. Serum SDF-1 levels were significantly elevated in HbSS/HbSβ0-thalassemia patients ([n=39], 3805 pg/mL (2121–6790)) as opposed to HbSC/HbSβ+-thalassemia patients ([n=18], 2405 pg/mL (1365–3047)) and healthy HbAA controls (n=25, 2623 pg/mL (2435–2988)) (p=0.04). During painful crisis SDF-1 levels increased significantly in both HbSS/HbSβ0-thalassemia ([n=24]) 26040 pg/mL (20135–27960)) and HbSC/HbSβ+-thalassemia patients ([n=5], 12666 pg/mL (3936–24453)) (p&lt;0.001 and p=0.01, respectively). Paired sample analysis was possible in 8 patients (SDF-1 determined in steady state and during painful crisis) and all patients demonstrated a strong SDF-1 increment from steady state levels (p=0.01) and serial measurements, available from 11 patients, showed persistently elevated SDF-1 levels well beyond crisis abatement (data not shown). Furthermore, SDF-1 levels were significantly higher in sickle cell patients with pulmonary hypertension as compared to patients without pulmonary hypertension in both HbSS/HbSβ0-thalassemia (PHT+ [n=8]: 6549 pg/mL (3893–7159), PHT- [n=24]: 2945 pg/mL (1560–6627)) and HbSC/HbSβ+-thalassemia (PHT+ [n=4] 3316 pg/mL (2900–6417) and PHT- [n=12] 2146 pg/mL (1138–2592)) patients (p=0.02 and p=0.01, respectively). SDF-1 levels were not related to other forms of organ damage, nor were they significantly associated with hemoglobin levels, the percentage of fetal hemoglobin or leukocyte counts in the clinically asymptomatic state (data not shown). Taken together, elevated circulating SDF-1 levels occur in patients with SCD and may play a role in the pathophysiology of acute and specific chronic disease related complications.

scholarly journals Patients with Sickle Cell Disease in Sicily Have Lower Rates of End Organ Damage, Allo-Immunization and Opioid Prescription Compared to a US Cohort

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3664-3664
Author(s):  
Caterina Minniti ◽  
Concetta Perrotta ◽  
Di Raimondo Francesco ◽  
Alessandra Quota ◽  
James G. Taylor
Keyword(s):  
Health Care ◽  
Disease Severity ◽  
Sickle Cell ◽  
Access To Health Care ◽  
African Ancestry ◽  
Organ Damage ◽  
Opioid Use ◽  
Clinical Complications ◽  
The Us ◽  
Organ Complications

Abstract Background: In developed countries, childhood mortality in sickle cell disease (SCD) is less than 5%, where the SCD morbidity and mortality burden has shifted to adults. As age increases, so do end organ complications like chronic pain. Environmental factors like health economics and genetic factors may influence such outcomes. Recent data from UK, showed higher than expected survival (ASH 2015) in the setting of a national health care system. The Hospital of Gela, Sicily has a well characterized Caucasian SCD population, which also has consistent access to health care via a nationalized system. We hypothesized that traditional disease severity markers including end organ damage, the rate of red blood cell (RBC) alloimmunization and opioid use would be lower in Sicily, compared to a modern US population of African ancestry with inconsistent access to healthcare. Methods: 90 SCD patients in Sicily were followed for over 9 years (2006-2014) to identify clinical complications, alloimmunization rates and survival. Demographics and sickle and alpha globin genotypes were documented. In the US, 632 SCD patients (excluding those with SC) had the same parameters collected as part of the Bethesda Sickle Cell Cohort Study. Laboratory and clinical complications were compared between populations according to phenotype groups (SS/SB0 or SB+ thalassemia). Clinical manifestations included hospitalizations for pain per year, RBC alloimmunization, hydroxyurea prescription rate. Statistical analysis utilized univariate comparisons (t and Chi square tests). Results: In Sicily, 51 patients had SS/SB0 and 28 SB+. All were of self-described Caucasian ancestry. No SC patients were identified in Sicily, therefore all comparisons were limited to SS/SB0 and SB+ thalassemia. Sicilian SS/SB0 patients were older than the US (p<0.0001), had lower AST (p=0.04) and creatinine (p=0.005), and higher HbF (p<0.0001). SB+ patients had similar characteristics (Table 1). Sixteen Sicilian patients (18%) died at a mean age of 53 years (range 31-77). As suggested by these survival data, SCD complications were less frequent for both SS/SB0 and SB+ groups in Sicily compared to the US (Table 2). More Sicilians were prescribed hydroxyurea, especially among those with SB+, although this was not a significant difference. The pain crisis hospitalizations per year was also higher in the US (p=0.008). Consistent with less frequent hospitalizations, only 4 of the Sicilian patients (3 SS/SB0, 1 SB+) were taking long acting opioids, compared to more frequent opioid use in the US. Finally, RBC alloimmunization, another surrogate measure for disease severity, was twice as common in SS/SB0 patients from the US (30%) as in Sicily (14%, p=0.01). Conclusions: These data suggest that end organ complications, like chronic pain and alloimmunization, in self-described Caucasians with uniform access to health care in Sicily, are less common than in a US cohort of adults. These differences could be attributable to access to specialized care, genetic differences in the proportion of African ancestry or environmental factors like more frequent use of preventative therapies like hydroxyurea. RBC alloimmunization in Sicily is significantly lower than reported in the US , but is still higher than observed in thalassemics in Italy. Perhaps this could be explained a matched genetic background between blood donors and recipients in Sicily, as has been suggested as a strategy to reduce RBC alloimmunization in the US. Further studies comparing unique SCD populations from different geographic regions may be helpful to elucidate genetic or environmental risk factors for end organ complications and disease severity. Table 1. Table 1. Table 2. Table 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sickle erythrocytes inhibit human endothelial cell DNA synthesis

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2146-2152 ◽  
Author(s):  
R Weinstein ◽  
MA Zhou ◽  
A Bartlett-Pandite ◽  
K Wenc
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Function ◽  
Dna Synthesis ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Inhibitory Effect ◽  
Human Endothelial Cell ◽  
Endothelial Monolayers ◽  
Sickle Erythrocytes

Abstract Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling. These results indicate that interaction between sickle erythrocytes and the endothelium may result in altered endothelial function. This altered endothelial function may contribute to the development of vascular occlusive phenomena in patients with sickle cell anemia.

scholarly journals An objective sign in painful crisis in sickle cell anemia: the concomitant reduction of high density red cells

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 559-563 ◽  
Author(s):  
ME Fabry ◽  
L Benjamin ◽  
C Lawrence ◽  
RL Nagel
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
State Level ◽  
Red Cells ◽  
Average Decrease ◽  
Concomitant Reduction ◽  
Objective Sign ◽  
The Stability ◽  
Painful Crisis

Abstract The etiopathologic basis of painful crisis in sickle cell anemia is largely unknown, and no objective criteria for its diagnosis and follow- up exist at present. We have studied 11 patients through 14 painful crises and observed a significant decrease of the densest fraction of red cells in 12 of the 14 crises as determined by isopycnic Percoll- Stractan continuous density gradients. If the first observation is normalized to 100%, the average decrease in dense cells was 77% with a range of 36% to 94%. The time needed for the percentage of dense cells to return to the steady-state level varied from seven to more than 30 days. These findings were in sharp contrast to the stability of the density pattern observed in another group of sickle cell patients, who were studied during crisis-free periods. The mechanism of the disappearance of dense cells could involve selective destruction by the reticuloendothelial (RE) system, selective sequestration in the areas of vasoocclusion, or a combination of both factors.

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