Contributions
Abstract: EP1200
Type: E-Poster Presentation
Session title: Sickle cell disease
Background
One of the most abundant proteins in the human body is Hemoglobin (Hb). Upon rupture of red blood cells, cell-free Hb may initiate adverse pathophysiological reactions. A well characterized example of Hb-triggered pathophysiological disorders is sickle cell disease (SCD). SCD is characterized by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where the generation of cytotoxic and pro-inflammatory reaction products are triggered. These processes eventually lead to endothelial cell activation and damage. The natural plasma protein hemopexin (Hpx) is a heme scavenger and binds heme in a 1:1 binding ratio with highest affinity. Hpx-bound heme is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation.
Aims
Investigating the protective function of Hpx on endothelial cells exposed to elevated levels of cell-free heme.
Methods
Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to heme in the presence or absence of different Hpx doses. As a read-out, different markers for endothelial cell activation were analyzed by Western Blot, multiplexed particle-based flow cytometry (Luminex), flow cytometry or on mRNA levels by qRT-PCR. Briefly, confluent HUVEC were preincubated with Hpx at different concentrations for 5 min before stimulation with heme for various lengths of time. Following stimulation cells were analyzed by Western Blot for the expression of HO-1, a sensitive marker for heme exposure. Expression of pro-inflammatory cytokines IL-6 and IL-8, cell adhesion molecule VCAM-1 and blood glycoprotein von Willebrand factor (vWF) were analyzed on mRNA levels by qRT-PCR or in cell culture supernatants by Luminex. All these investigated proteins are well known markers for endothelial cell activation.
Alternatively, heme stimulation of Hpx-preincubated HUVEC was conducted for 25 min and cells were analyzed by flow cytometry for membrane bound P-Selectin expression, another robust endothelial cell activation marker.
Results
In the absence of Hpx, Heme consistently showed strong stimulatory capacity on HUVEC reflected in a robust upregulation of pro-inflammatory cytokines, VCAM-1, vWF, HO-1 and P-Selectin. Increasing Hpx concentrations during heme-mediated stimulation led to a dose-dependent reduction in HUVEC activation markers and once an equimolar ratio between heme and Hpx had been reached, the expression of HO-1, P-Selectin, VCAM-1, vWF and pro-inflammatory cytokines was lowered to background levels.
Conclusion
The presented data underlines on the one hand the stimulatory capacity of heme on endothelial cells and demonstrates on the other hand the efficient heme scavenging potential of Hpx. Hpx-bound heme molecules are rendered non-reactive and consequently, Hpx potently prevents the pro-inflammatory effect of heme on endothelial cells. This effect results in a strongly reduced expression of endothelial activation markers in heme-stimulated cells under an equimolar ratio of heme and Hpx. Hence, our study suggests a protective role of Hpx for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis, a phenomenon seen in Hb-triggered pathophysiological disorders such as SCD.
Keyword(s): Endothelial cell, Heme, Sickle cell disease
Abstract: EP1200
Type: E-Poster Presentation
Session title: Sickle cell disease
Background
One of the most abundant proteins in the human body is Hemoglobin (Hb). Upon rupture of red blood cells, cell-free Hb may initiate adverse pathophysiological reactions. A well characterized example of Hb-triggered pathophysiological disorders is sickle cell disease (SCD). SCD is characterized by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where the generation of cytotoxic and pro-inflammatory reaction products are triggered. These processes eventually lead to endothelial cell activation and damage. The natural plasma protein hemopexin (Hpx) is a heme scavenger and binds heme in a 1:1 binding ratio with highest affinity. Hpx-bound heme is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation.
Aims
Investigating the protective function of Hpx on endothelial cells exposed to elevated levels of cell-free heme.
Methods
Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to heme in the presence or absence of different Hpx doses. As a read-out, different markers for endothelial cell activation were analyzed by Western Blot, multiplexed particle-based flow cytometry (Luminex), flow cytometry or on mRNA levels by qRT-PCR. Briefly, confluent HUVEC were preincubated with Hpx at different concentrations for 5 min before stimulation with heme for various lengths of time. Following stimulation cells were analyzed by Western Blot for the expression of HO-1, a sensitive marker for heme exposure. Expression of pro-inflammatory cytokines IL-6 and IL-8, cell adhesion molecule VCAM-1 and blood glycoprotein von Willebrand factor (vWF) were analyzed on mRNA levels by qRT-PCR or in cell culture supernatants by Luminex. All these investigated proteins are well known markers for endothelial cell activation.
Alternatively, heme stimulation of Hpx-preincubated HUVEC was conducted for 25 min and cells were analyzed by flow cytometry for membrane bound P-Selectin expression, another robust endothelial cell activation marker.
Results
In the absence of Hpx, Heme consistently showed strong stimulatory capacity on HUVEC reflected in a robust upregulation of pro-inflammatory cytokines, VCAM-1, vWF, HO-1 and P-Selectin. Increasing Hpx concentrations during heme-mediated stimulation led to a dose-dependent reduction in HUVEC activation markers and once an equimolar ratio between heme and Hpx had been reached, the expression of HO-1, P-Selectin, VCAM-1, vWF and pro-inflammatory cytokines was lowered to background levels.
Conclusion
The presented data underlines on the one hand the stimulatory capacity of heme on endothelial cells and demonstrates on the other hand the efficient heme scavenging potential of Hpx. Hpx-bound heme molecules are rendered non-reactive and consequently, Hpx potently prevents the pro-inflammatory effect of heme on endothelial cells. This effect results in a strongly reduced expression of endothelial activation markers in heme-stimulated cells under an equimolar ratio of heme and Hpx. Hence, our study suggests a protective role of Hpx for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis, a phenomenon seen in Hb-triggered pathophysiological disorders such as SCD.
Keyword(s): Endothelial cell, Heme, Sickle cell disease