Protective Role of HO-1 Expressing CD16+ Patrolling Monocytes Against Hemolysis-Induced Endothelial Damage and Vaso-Occlusive Crisis in Sickle Cell Disease

Abstract Causing leukocyte activation and upregulation of adhesion molecules on endothelial cells. CD16+ monocytes, also known as endothelial patrolling monocytes, normally scavenge the damaged cells and debris from the vasculature. As compared to other monocyte subsets or immune cell types, the CD16+monocyte subset expresses higher levels of the anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Given the role of CD16+ monocytes as scavengers of debris on endothelial cells, we tested the hypothesis that this subset may protect SCD vasculature from the ongoing hemolytic insult through expression of high levels of HO-1. We found roughly 35% of circulating CD16+ monocytes from SCD patients expressed very high levels of HO-1 as compared to 5% in healthy controls. The HO-1hi SCD monocytes expressed significantly (30%) less TNF-a compared to HO-1lo monocytes following stimulation, consistent with anti-inflammatory effects of HO-1. We hypothesized that uptake of free hemoglobin/heme was responsible for high HO-1 expression levels in SCD CD16+ monocytes. To test this, healthy donors (HDs) or SCD patient monocytes were treated with different doses of free heme or hemolysed RBCs. We found dose-dependent HO-1 induction (five-fold at 20mM heme) in purified CD16- monocytes, but surprisingly none in CD16+ subset. However, upon co-culture with human umbilical vein endothelial cells (HUVEC), continuous or prior exposure to heme induced HO-1hi expression exclusively in CD16+ monocytes (5 fold in HD and further two fold in SCD compared to non-heme treated cocultures, p<0.001). Using imagining flow cytometric analysis, we found marked increase in uptake of heme-exposed endothelial cell-derived material by CD16+ monocytes (HD: 2% to 13% ± 3%; in SCD: 20% ± 3% to 30% ± 4%, p< 0.001) but none by CD16- monocytes. Our transwell studies demonstrated that cell-cell contact between CD16+ monocytes and heme-exposed HUVEC was required for HO-1hi expression. We found roughly 4-fold increase in expression of phosphatidylserine (PS, annexin V+), ICAM-1 and vCAM-1 on heme-treated HUVEC cells. Antibody blocking studies identified PS moieties as well as ICAM-1 as key molecules involved in monocyte-HUVEC interactions that mediated HO-1hi induction, suggesting that high levels of HO-1 expression in SCD CD16+ monocytes is in part the result of attachment to and engulfment of apoptotic, activated endothelial cells damaged by heme. SCD patients suffer from vaso-occlusive crisis (VOC), resulting from increased attachment of SCD RBCs to damaged and activated endothelium. We hypothesized that inadequate numbers or lower HO-1hi levels in CD16+ monocyte will predispose SCD patients to episodes of VOC due to decreased removal by CD16+ monocytes of damaged endothelial and sickle RBCs. Amongst SCD patients receiving chronic transfusions, we found a two-fold lower frequency of circulating CD16+ monocytes and half the numbers of CD16+HO-1hi monocytes in patients with a recent history of VOC episode as compared to those without VOC (p< 0.01); the former group also expressed higher levels of circulating sVCAM-1 (997 ± 210 vs 765 ± 236 ng/m, p=0.02), a marker of endothelial activation. To formally test the role of patrolling monocytes in endothelial damage induced by SCD RBCs and heme, we injected RBCs from Townes SCD mice alone or after 24hrs with heme into Nr4a1-knockout mice which have a selective loss of patrolling monocytes. Immunofluorescence analysis of liver vasculature showed a 3 fold increase in the activated endothelial marker, ICAM-1 within 24hrs following injection of sickle RBCs and two-fold increase in circulating sVCAM-1 levels in mice treated with sickle RBC plus heme (p <0.001). Transfer of HO-1+ patrolling monocytes (LY6Clo), but not a classical monocyte subset (LY6C+) reversed activated endothelial phenotype, indicating that patrolling monocytes can inhibit SCD-induced endothelial activation. Altogether, these data suggest that SCD patrolling monocytes remove hemolysis-damaged endothelial cells, resulting in HO-1 upregulation and dampening of vascular inflammation. Perturbations in CD16+ monocyte numbers resulting in lower local HO-1 levels can predispose SCD patients to VOC, thus identifying HO-1+ patrolling monocytes as key players in VOC pathophysiology and as therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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Anti-Inflammatory and Antiatherogenic Role of BMP Receptor II in Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.112.300287 ◽

2013 ◽

Vol 33 (6) ◽

pp. 1350-1359 ◽

Cited By ~ 48

Author(s):

Chan Woo Kim ◽

Hannah Song ◽

Sandeep Kumar ◽

Douglas Nam ◽

Hyuk Sang Kwon ◽

...

Keyword(s):

Endothelial Cells ◽

Bmp Receptor ◽

Anti Inflammatory

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Role of nuclear factor-κ B and heme oxygenase-1 in the mechanism of action of an anti-inflammatory chalcone derivative in RAW 264.7 cells

British Journal of Pharmacology ◽

10.1038/sj.bjp.0705821 ◽

2004 ◽

Vol 142 (7) ◽

pp. 1191-1199 ◽

Cited By ~ 59

Author(s):

María José Alcaraz ◽

Ana María Vicente ◽

Amparo Araico ◽

José N Dominguez ◽

María Carmen Terencio ◽

...

Keyword(s):

Heme Oxygenase ◽

Mechanism Of Action ◽

Raw 264.7 Cells ◽

Heme Oxygenase 1 ◽

Raw 264.7 ◽

Nuclear Factor ◽

Chalcone Derivative ◽

Anti Inflammatory ◽

Nuclear Factor Κ B

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Abstract TP99: Human Lung Endothelial Cell Anti-Inflammatory and Regenerative Responses in Acute Ischemic Stroke

Stroke ◽

10.1161/str.51.suppl_1.tp99 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Nikunj Satani ◽

Kaavya Giridhar ◽

Natalia Wewior ◽

Dominique D Norris ◽

Scott D Olson ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Inflammatory Responses ◽

Inflammatory Factors ◽

Stroke Severity ◽

Stroke Patients ◽

Anti Inflammatory ◽

Lung Endothelial Cells ◽

Stroke Mimic

Background: Inflammatory responses after stroke consists of central and peripheral immune responses. The role of the spleen after stroke is well-known, however the role of the lungs has not been studied in detail. We explored the relation between stroke severity and immunomodulatory changes in lung endothelial cells. Methods: Human pulmonary endothelial cells (hPECs, Cell Biologics) were cultured at passage 3. Serum from stroke patients with NIH Stroke Scale (NIHSS) severity ranging from 0 to 20 was collected at 24 hours after stroke. hPECs were exposed to media with 1) 10% FBS alone (N=6), 2) 10% serum from stroke patients (N=72), or 3) 10% serum from stroke mimic patients (N=6). After 3 hour of exposure, fresh media was added and secretomes from hPECs were measured after 24 hours. We isolated RNA from hPECs after 3 hour of serum exposure and measured gene expression (N=6 for each group). Secretome and gene changes in hPECs were analyzed based on stroke severity, tPA treatment, and co-morbidities. Results: Serum from stroke patients reduced the secretion of IL-8, MCP-1 and Fractalkine (p<0.01), and increased the secretion of VEGF and BDNF (p<0.01) from hPECs. These effects were more pronounced depending on stroke severity (Fig). There was no effect of tPA or T2DM on hPECs secretomes. There was significantly reduced gene expression of IL-6, IL-8, MCP-1 and IL-1β and significantly higher expression of ICAM1, IGF-1 and TGF-β1 as compared to stroke mimics. Conclusion: Exposure of hPECs to serum from stroke patients alters their immunomodulatory properties. Higher severity of stroke leads to more protective response from hPECs by reducing the secretion of pro-inflammatory factors, while increasing the secretion of anti-inflammatory factors.

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Effect of Blueberry Anthocyanins Malvidin and Glycosides on the Antioxidant Properties in Endothelial Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/1591803 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 31

Author(s):

Wuyang Huang ◽

Yunming Zhu ◽

Chunyang Li ◽

Zhongquan Sui ◽

Weihong Min

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Endothelial Cells ◽

Functional Food ◽

Functional Role ◽

Antioxidant Properties ◽

Heme Oxygenase 1 ◽

Oxygen Species ◽

Good Resource

The objective of this research was to survey the antioxidant functional role of the main anthocyanins of blueberries in endothelial cells. Changes on the reactive oxygen species (ROS), xanthine oxidase-1 (XO-1), superoxide dismutase (SOD), and heme oxygenase-1 (HO-1) in cells of malvidin and the two glycosides were investigated. The results showed that these anthocyanins decreased the levels of ROS and XO-1 but increased the levels of SOD and HO-1. Glycosides improved the antioxidant capacity of malvidin to a great extent. The changes in the antioxidant properties of malvidin-3-glucoside were more pronounced than malvidin-3-galactoside. Variation in levels of malvidin-3-glucoside and malvidin-3-galactoside had a significant impact on antioxidant properties to different extents. It indicates that blueberries are a good resource of anthocyanins, which can protect cells from oxidative deterioration and use blueberry as a potential functional food to prevent diseases related to oxidative stress.

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Endothelial Activation by Sickle Mouse Red Cells and Their Endothelial Adhesion In Vivo is Abolished by Catalase Treatment of Sickle Cells, but Not Quiescent Endothelium, Implying a Role of Heme-Mediated Peroxide Generation in Sickle Cell Adhesion.

Blood ◽

10.1182/blood.v114.22.902.902 ◽

2009 ◽

Vol 114 (22) ◽

pp. 902-902 ◽

Cited By ~ 1

Author(s):

Dhananjay K. Kaul ◽

Sandra M. Suzuka ◽

Mary Fabry

Keyword(s):

Cell Adhesion ◽

Adhesion Molecules ◽

Blood Cells ◽

Fold Increase ◽

Endothelial Activation ◽

Red Cells ◽

Time Dependent ◽

C57bl Mouse ◽

C57bl Mice

Abstract Abstract 902 Multiple adhesion molecules, expressed on sickle red blood cells (SS RBCs) and activated endothelium, have been implicated in SS RBC adhesion to vascular endothelium. Moreover, intrinsic differences among heterogeneous SS RBC subpopulations, involving differences in red cell adhesion molecules and cell deformability, may contribute to their adhesive and obstructive properties and lead to postcapillary obstruction. However, the role of SS RBCs in endothelium activation and adhesion has not been evaluated despite the insightful studies of Hebbel and coworkers (JCI, 1982) demonstrating that SS RBCs generate excessive amounts of reactive oxygen species due to the presence of unstable hemoglobin S (HbS) and autoxidation of iron in heme. RBCs from transgenic-knockout sickle (BERK) mice similarly show a pronounced increase in heme degradation (Nagababu et. al. Blood Cells Mol Dis, 2008). We hypothesize that hypoxic conditions in venules (oxygen tension,∼30 mm Hg) will accelerate autoxidation of RBC membrane-bound HbS and release H2O2 that will be transferred to adjoining endothelium resulting in its activation (i.e., up-regulation of endothelial adhesion molecules) and SS RBC adhesion. To test the hypothesis that HbS-containing red cells from BERK mice will result in activation of quiescent endothelium in normal mice, we infused FITC (fluorescein isothiocynate)-labeled BERK red cells into congenic C57BL mice. BERK mice, expressing exclusively human βS- and α-globins, have been extensively backcrossed onto C57BL background. Intravital observations were made in the cremaster muscle microcirculatory bed. A single bolus of 150 μl of FITC-labeled BERK RBCs (Hct 30%) was infused into the recipient C57BL mouse via the jugular vein over a period of 5 min to avoid any shear related platelet aggregation. Infusion of FITC-labeled control (C57BL) mouse RBCs into C57BL recipient mice resulted in rare or no RBC adhesion, suggesting that there was no activating effect on endothelium. In contrast, infusion of BERK mouse RBCs into C57BL mice resulted in time-dependent increase in adhesion to venular endothelium. Adhesion became discernable after 3 minutes and showed a 3-5 fold increase after 5-min compared with the number of adherent RBCs at 3 min (P<0.01). Next, we investigated if the infusion of BERK mouse RBCs would induce increased endothelial oxidants. To this end, the cremaster preparation was suffused for 15 min with 123 dihydrorhodamine (DHR), a H2O2-sensitive probe (10 μl/L), followed by a bolus infusion of BERK mouse RBCs, and time-dependent changes in DHR fluorescence intensity were monitored in venules, the sites of adhesion. Infusion of BERK mouse RBCs, but not C57BL RBCs, resulted in time-dependent increase in the fluorescence intensity (ΔI) in venular endothelium, with almost 5-fold increase in DHR intensity after 5 min of BERK RBC infusion (P<0.001) compared with ΔI at 1 min. When infusion of catalase (900 U/mouse) into recipient C57BL mice was followed 30 min later by a bolus of FITC-labeled BERK mouse RBCs, BERK RBC adhesion and pronounced DHR fluorescence in endothelium were observed, demonstrating that intravascular infusion of catalase had little effect on oxidant generation by BERK mouse RBCs. In contrast, infusion of BERK RBCs pre-treated with catalase (100 U in 0.2 ml RBC suspension, 9-fold less catalase per mouse) to quench RBC generated H2O2 inhibited endothelial DHR fluorescence and BERK RBC adhesion. These results strongly suggest an obligatory role of heme-mediated peroxide generation by SS RBC in endothelial activation and SS RBC adhesion, and support the notion that heme-mediated oxidant generation may play a vital role in endothelial dysfunction in sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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Direct evidence of endothelial injury during cardiopulmonary bypass by demonstration of circulating endothelial cells

Perfusion ◽

10.1191/0267659106pf860oa ◽

2006 ◽

Vol 21 (3) ◽

pp. 133-137 ◽

Cited By ~ 11

Author(s):

Franz-Xaver Schmid ◽

Bernhard Floerchinger ◽

Nalini Kumar Vudattu ◽

Günther Eissner ◽

Marion Haubitz ◽

...

Keyword(s):

Endothelial Cells ◽

Cardiopulmonary Bypass ◽

Direct Evidence ◽

Artery Bypass ◽

Endothelial Activation ◽

Endothelial Damage ◽

Endothelial Injury ◽

Circulating Endothelial Cells ◽

Whole Body ◽

Maximum Increase

Endothelial activation is considered a key process in the development of a whole body inflammatory response secondary to cardiopulmonary bypass (CPB). Increased levels of a multitude of soluble mediators have been described as being released during and after cardiac surgery. Circulating endothelial cells have recently been established as a novel marker of endothelial damage in a variety of vascular disorders. Blood samples from 20 patients undergoing elective coronary artery bypass surgery were obtained preoperatively and 1, 6, 12, 24, and 48 h after termination of CPB. Control samples were obtained from ten healthy volunteers. Circulating endothelial cells (CEC) were isolated with immunomagnetic anti-CD146-coated Dynabeads, and counted in a Nageotte chamber. Low numbers of CEC were observed in healthy control volunteers (12±6 cells/mL; median: 9 cells/mL). CEC numbers were already significantly elevated in all patients before CPB, and there was a further significant increase after weaning from CPB (maximum increase at 6 h after CPB: 73±30 cells/mL; range: 30-153 cells/mL, p < 0.001). The number of CEC provides further and direct evidence that CPB is associated with a pronounced endothelial injury and/or damage. CEC appear to be most useful markers for vascular endothelial activation because they are specific, stable, and circulating components of injured vessel wall.

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CD97/ADGRE5 Inhibits LPS Induced NF-κB Activation through PPAR-γUpregulation in Macrophages

Mediators of Inflammation ◽

10.1155/2016/1605948 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Shuai Wang ◽

Zewei Sun ◽

Wenting Zhao ◽

Zhen Wang ◽

Mingjie Wu ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Immunofluorescence Staining ◽

Inflammatory Factor ◽

Negative Role ◽

Anti Inflammatory

CD97/ADGRE5 protein is predominantly expressed on leukocytes and belongs to the EGF-TM7 receptors family. It mediates granulocytes accumulation in the inflammatory tissues and is involved in firm adhesion of PMNC on activated endothelial cells. There have not been any studies exploring the role of CD97 in LPS induced NF-κB activation in macrophages. Therefore, we first measured the CD97 expression in LPS treated human primary macrophages and subsequently analyzed the levels of inflammatory factor TNF-αand transcription factor NF-κB in these macrophages that have been manipulated with either CD97 knockdown or overexpression. We found that a reported anti-inflammatory transcription factor, PPAR-γ, was involved in the CD97 mediated NF-κB suppression. Furthermore, by immunofluorescence staining, we established that CD97 overexpression not only inhibited LPS induced p65 expression in the nucleus but also promoted the PPAR-γexpression. Moreover, using CD97 knockout THP-1 cells, we further demonstrated that CD97 promoted PPAR-γexpression and decreased LPS induced NF-κB activation. In conclusion, CD97 plays a negative role in LPS induced NF-κB activation and TNF-αsecretion, partly through PPAR-γupregulation.

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Acute moderate-intensity exercise in middle-aged men has neither an anti- nor proinflammatory effect

Journal of Applied Physiology ◽

10.1152/japplphysiol.00096.2008 ◽

2008 ◽

Vol 105 (1) ◽

pp. 260-265 ◽

Cited By ~ 39

Author(s):

Daniella Markovitch ◽

Rex M. Tyrrell ◽

Dylan Thompson

Keyword(s):

Physical Activity ◽

Endothelial Cells ◽

Inflammatory Response ◽

Moderate Intensity ◽

Heme Oxygenase 1 ◽

Moderate Intensity Exercise ◽

Moderate Intensity Physical Activity ◽

Cultured Endothelial Cells ◽

Intensity Physical Activity ◽

Anti Inflammatory

Strenuous exercise induces an initial pro- and subsequent anti-inflammatory response, and it has been suggested that this may be one of the ways that regular exercise reduces chronic inflammation and therefore the risk of cardiovascular disease. However, public health recommendations emphasize moderate-intensity physical activity, and it is important to understand whether moderate-intensity exercise has a similar anti-inflammatory effect. Twelve sedentary male volunteers (age 54 ± 4 yr) completed two main trials, moderate-intensity exercise and rest (30 min at 50% maximal oxygen uptake vs. sitting, respectively). There were no significant changes in circulating neutrophils, lymphocytes, monocytes, or serum interleukin-6, interleukin-10, and C-reactive protein concentration over the 7 days following exercise. Similarly, lymphocyte adhesion to cultured endothelial cells and heme oxygenase-1 (HO-1) expression in lymphocytes and monocytes were not affected by walking at any time point. These results suggest that the long-term anti-inflammatory and antiatherogenic effects of regular moderate-intensity physical activity must be explained by something other than a profound net anti-inflammatory response to each exercise bout since a single bout of walking did not lead to a change in various markers of inflammation or lymphocyte adherence to cultured endothelial cells.

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