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Platelet Vascular Endothelial Growth Factor is a Potential Mediator of Transfusion-Related Acute Lung Injury.

Maloney JP, Ambruso DR, Voelkel NF, Silliman CC - J Pulm Respir Med (2014)

Bottom Line: Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak.Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels.At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pulmonary Sciences and Critical Care Medicine, University of Colorado at Denver, USA ; Department of Medicine, University of Colorado at Denver, USA.

ABSTRACT

Objective: The occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors.

Methods: Single donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak.

Results: There was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, p<0.01, n=8) and a contribution by contaminating leukocytes was excluded. Exogenous 125I-VEGF bound avidly and specifically to the lung vasculature, and unlabeled VEGF in the lung perfusate caused vascular leak.

Conclusion: Rising concentrations of VEGF occur during storage of single donor platelet concentrates due to platelet secretion or disintegration, but not due to leukocyte contamination. Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels. At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs. These data provide further evidence that VEGF may contribute to the increased lung permeability seen in TRALI associated with platelet products.

No MeSH data available.


Related in: MedlinePlus

Lung binding of 125I-VEGF added to the perfusate of isolated perfused rat lungs (IPRL), as determined by counts per minute (CPM) of radioactivity per gram of lung tissue. The final [125I-VEGF] was 1 pM (23 pg/ml), similar to the [VEGF] of platelet supernatants in this study. 125I-VEGF bound avidly to VEGF receptors within the pulmonary vasculature (Vehicle, pre-incubation of 125I-VEGF with 10 ul of PBS). This binding amounted to 30–40% of added radioactivity and was specific to VEGF receptors as it was potently inhibited by pre-perfusion with a 1000-fold excess concentration of unlabeled (“Cold”) VEGF and was strongly inhibited when 125I-VEGF was pre-incubated with a VEGF antibody (VEGF-Ab in 10 ul of PBS), whereas a control antibody (CMV-Ab in 10 ul of PBS) had no significant effect. Perfusion with 125I-VEGF was done for 15 minutes. * P < 0.0001 by unpaired t-test. Results are depicted as group mean ± SEM
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Figure 3: Lung binding of 125I-VEGF added to the perfusate of isolated perfused rat lungs (IPRL), as determined by counts per minute (CPM) of radioactivity per gram of lung tissue. The final [125I-VEGF] was 1 pM (23 pg/ml), similar to the [VEGF] of platelet supernatants in this study. 125I-VEGF bound avidly to VEGF receptors within the pulmonary vasculature (Vehicle, pre-incubation of 125I-VEGF with 10 ul of PBS). This binding amounted to 30–40% of added radioactivity and was specific to VEGF receptors as it was potently inhibited by pre-perfusion with a 1000-fold excess concentration of unlabeled (“Cold”) VEGF and was strongly inhibited when 125I-VEGF was pre-incubated with a VEGF antibody (VEGF-Ab in 10 ul of PBS), whereas a control antibody (CMV-Ab in 10 ul of PBS) had no significant effect. Perfusion with 125I-VEGF was done for 15 minutes. * P < 0.0001 by unpaired t-test. Results are depicted as group mean ± SEM

Mentions: 125I-VEGF (1 pM, or 23 pg/ml) added to the lung perfusion solution bound avidly and specifically to VEGF receptors within the pulmonary microvasculature. This 125I-VEGF binding under control conditions (PBS pre-incubation with 125I-VEGF) amounted to 30–40% of added radioactivity (about 20% of the initial radioactivity remained in perfusate at the end of experiments, the rest of the radioactivity appeared mostly absorbed onto plastic surfaces of tubing and the buffer reservoir). Binding of VEGF within hearts was minimal, being 1.81 ± 2.19% (mean ± SEM) of lung binding. Binding was specific for endothelial VEGF receptors as it was inhibited nearly completely (to 10.5% of vehicle control) by a 1000-fold (1 nM) excess concentration of unlabeled or “cold” VEGF in the perfusate (vehicle control 43,115 ± 994 cpm/gram wet lung; “cold” VEGF 4,517 ± 133 cpm/gram wet lung, P < .0001; mean ± SEM; Figure 3). Moreover, binding was further shown to be specific as it was inhibited by 55% when 125I-VEGF was pre-incubated with an anti-VEGF antibody, whereas binding was not inhibited by pre-incubation with an antibody of the same isotype that binds an unrelated target (anti-CMV antibody 40,489 ± 2,512 cpm/gram wet lung; anti-VEGF antibody 18,455 ± 1,853 cpm/gram wet lung, P < .0001; mean ± SEM; Figure 3).


Platelet Vascular Endothelial Growth Factor is a Potential Mediator of Transfusion-Related Acute Lung Injury.

Maloney JP, Ambruso DR, Voelkel NF, Silliman CC - J Pulm Respir Med (2014)

Lung binding of 125I-VEGF added to the perfusate of isolated perfused rat lungs (IPRL), as determined by counts per minute (CPM) of radioactivity per gram of lung tissue. The final [125I-VEGF] was 1 pM (23 pg/ml), similar to the [VEGF] of platelet supernatants in this study. 125I-VEGF bound avidly to VEGF receptors within the pulmonary vasculature (Vehicle, pre-incubation of 125I-VEGF with 10 ul of PBS). This binding amounted to 30–40% of added radioactivity and was specific to VEGF receptors as it was potently inhibited by pre-perfusion with a 1000-fold excess concentration of unlabeled (“Cold”) VEGF and was strongly inhibited when 125I-VEGF was pre-incubated with a VEGF antibody (VEGF-Ab in 10 ul of PBS), whereas a control antibody (CMV-Ab in 10 ul of PBS) had no significant effect. Perfusion with 125I-VEGF was done for 15 minutes. * P < 0.0001 by unpaired t-test. Results are depicted as group mean ± SEM
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4335657&req=5

Figure 3: Lung binding of 125I-VEGF added to the perfusate of isolated perfused rat lungs (IPRL), as determined by counts per minute (CPM) of radioactivity per gram of lung tissue. The final [125I-VEGF] was 1 pM (23 pg/ml), similar to the [VEGF] of platelet supernatants in this study. 125I-VEGF bound avidly to VEGF receptors within the pulmonary vasculature (Vehicle, pre-incubation of 125I-VEGF with 10 ul of PBS). This binding amounted to 30–40% of added radioactivity and was specific to VEGF receptors as it was potently inhibited by pre-perfusion with a 1000-fold excess concentration of unlabeled (“Cold”) VEGF and was strongly inhibited when 125I-VEGF was pre-incubated with a VEGF antibody (VEGF-Ab in 10 ul of PBS), whereas a control antibody (CMV-Ab in 10 ul of PBS) had no significant effect. Perfusion with 125I-VEGF was done for 15 minutes. * P < 0.0001 by unpaired t-test. Results are depicted as group mean ± SEM
Mentions: 125I-VEGF (1 pM, or 23 pg/ml) added to the lung perfusion solution bound avidly and specifically to VEGF receptors within the pulmonary microvasculature. This 125I-VEGF binding under control conditions (PBS pre-incubation with 125I-VEGF) amounted to 30–40% of added radioactivity (about 20% of the initial radioactivity remained in perfusate at the end of experiments, the rest of the radioactivity appeared mostly absorbed onto plastic surfaces of tubing and the buffer reservoir). Binding of VEGF within hearts was minimal, being 1.81 ± 2.19% (mean ± SEM) of lung binding. Binding was specific for endothelial VEGF receptors as it was inhibited nearly completely (to 10.5% of vehicle control) by a 1000-fold (1 nM) excess concentration of unlabeled or “cold” VEGF in the perfusate (vehicle control 43,115 ± 994 cpm/gram wet lung; “cold” VEGF 4,517 ± 133 cpm/gram wet lung, P < .0001; mean ± SEM; Figure 3). Moreover, binding was further shown to be specific as it was inhibited by 55% when 125I-VEGF was pre-incubated with an anti-VEGF antibody, whereas binding was not inhibited by pre-incubation with an antibody of the same isotype that binds an unrelated target (anti-CMV antibody 40,489 ± 2,512 cpm/gram wet lung; anti-VEGF antibody 18,455 ± 1,853 cpm/gram wet lung, P < .0001; mean ± SEM; Figure 3).

Bottom Line: Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak.Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels.At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pulmonary Sciences and Critical Care Medicine, University of Colorado at Denver, USA ; Department of Medicine, University of Colorado at Denver, USA.

ABSTRACT

Objective: The occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors.

Methods: Single donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak.

Results: There was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, p<0.01, n=8) and a contribution by contaminating leukocytes was excluded. Exogenous 125I-VEGF bound avidly and specifically to the lung vasculature, and unlabeled VEGF in the lung perfusate caused vascular leak.

Conclusion: Rising concentrations of VEGF occur during storage of single donor platelet concentrates due to platelet secretion or disintegration, but not due to leukocyte contamination. Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels. At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs. These data provide further evidence that VEGF may contribute to the increased lung permeability seen in TRALI associated with platelet products.

No MeSH data available.


Related in: MedlinePlus