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Complement Mediates Von Willebrand Factor Release in Obstructive Sleep Apnea
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A6410 - Complement Mediates Von Willebrand Factor Release in Obstructive Sleep Apnea
Author Block: T. Thoma1, M. Emin2, S. Gao2, R. Shah2, S. Jelic2; 1Division of Pediatric Pulmonology, Columbia University College of Physicians and Surgeon's, New York, NY, United States, 2Division of Pulmonary, Allergy and Critical Care Medicine, Columbia University College of Physicians and Surgeon's, New York, NY, United States.
RATIONALE: Obstructive sleep apnea (OSA) is associated with increased risk for arterial and venous thromboembolic events; however, the underlying mechanisms are unclear. Complement inhibitor CD59 is internalized from the endothelial cell (EC) membrane in OSA, thereby reducing protection against complement. Surprisingly, intracellular CD59 colocalizes with von Willebrand factor (vWF) in ECs of OSA patients. We hypothesized that decreased endothelial complement inhibition promotes vWF release in intermittent hypoxia (IH), a hallmark of OSA.
METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to 8 h of IH (alternating 30 min normoxia/30 min hypoxia) or normoxia while incubated with heat-inactivated (complement-free) or 20% normal human serum (contains complement). vWF in the culture media was quantified using Western blotting. To assess potential role of individual complement components in vWF release, human recombinant C9 and C6 were added separately to inactivated serum and vWF release was measured again. Individual interactions between vWF and C9 were assessed using immunofluorescence and confocal microscopy.
RESULTS: Levels of vWF in culture media were greater in IH compared with normoxia after incubation with complement-containing serum whereas its levels were similar after incubation with complement-free serum (mean grey level [SD] 75±11 vs. 52±22, p=0.02 and 70±20 vs. 74±13, p=NS, respectively, n=10). Addition of C9 but not C6 (p=NS) to the complement-free serum increased vWF levels in culture media in IH compared to normoxia (mean grey level [SD] 42±10 vs. 39±8, p=0.04, n=5) whereas the percent of intracellular vWF colocalized with C9 increased in IH compared to normoxia (mean% [SD] 0.22±0.07 vs 0.11±0.03, p=0.04, n=4), suggesting a role for C9 in vWF release.
CONCLUSIONS: IH promotes release of vWF from ECs in complement-dependent manner. Decreased complement inhibition may contribute to prothrombotic condition in OSA.
Author Block: T. Thoma1, M. Emin2, S. Gao2, R. Shah2, S. Jelic2; 1Division of Pediatric Pulmonology, Columbia University College of Physicians and Surgeon's, New York, NY, United States, 2Division of Pulmonary, Allergy and Critical Care Medicine, Columbia University College of Physicians and Surgeon's, New York, NY, United States.
RATIONALE: Obstructive sleep apnea (OSA) is associated with increased risk for arterial and venous thromboembolic events; however, the underlying mechanisms are unclear. Complement inhibitor CD59 is internalized from the endothelial cell (EC) membrane in OSA, thereby reducing protection against complement. Surprisingly, intracellular CD59 colocalizes with von Willebrand factor (vWF) in ECs of OSA patients. We hypothesized that decreased endothelial complement inhibition promotes vWF release in intermittent hypoxia (IH), a hallmark of OSA.
METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to 8 h of IH (alternating 30 min normoxia/30 min hypoxia) or normoxia while incubated with heat-inactivated (complement-free) or 20% normal human serum (contains complement). vWF in the culture media was quantified using Western blotting. To assess potential role of individual complement components in vWF release, human recombinant C9 and C6 were added separately to inactivated serum and vWF release was measured again. Individual interactions between vWF and C9 were assessed using immunofluorescence and confocal microscopy.
RESULTS: Levels of vWF in culture media were greater in IH compared with normoxia after incubation with complement-containing serum whereas its levels were similar after incubation with complement-free serum (mean grey level [SD] 75±11 vs. 52±22, p=0.02 and 70±20 vs. 74±13, p=NS, respectively, n=10). Addition of C9 but not C6 (p=NS) to the complement-free serum increased vWF levels in culture media in IH compared to normoxia (mean grey level [SD] 42±10 vs. 39±8, p=0.04, n=5) whereas the percent of intracellular vWF colocalized with C9 increased in IH compared to normoxia (mean% [SD] 0.22±0.07 vs 0.11±0.03, p=0.04, n=4), suggesting a role for C9 in vWF release.
CONCLUSIONS: IH promotes release of vWF from ECs in complement-dependent manner. Decreased complement inhibition may contribute to prothrombotic condition in OSA.
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