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Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

Turner NA, Moake JL - PLoS ONE (2015)

Bottom Line: In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs.The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H.Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Rice University, Houston, Texas, United States of America.

ABSTRACT
The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

No MeSH data available.


Related in: MedlinePlus

Statistical confirmation of the presence of FVIII and VWF in WPBs.GMVECs and HUVECs were treated with Triton-X to allow internal staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse IgG AF-647 (red) followed by staining with rabbit anti-human VWF plus chicken anti-rabbit IgG AF-488 (green). Merged images of FVIII and VWF detection, along with the corresponding intensity scatter plot, are shown in: (A and C) GMVECs at 60×, N = 4; (B and D) GMVECs at 100×, N = 5; (E and G) HUVECs at 60×, N = 7; and (F and H) HUVECs at 100×, N = 7. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.
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pone.0140740.g005: Statistical confirmation of the presence of FVIII and VWF in WPBs.GMVECs and HUVECs were treated with Triton-X to allow internal staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse IgG AF-647 (red) followed by staining with rabbit anti-human VWF plus chicken anti-rabbit IgG AF-488 (green). Merged images of FVIII and VWF detection, along with the corresponding intensity scatter plot, are shown in: (A and C) GMVECs at 60×, N = 4; (B and D) GMVECs at 100×, N = 5; (E and G) HUVECs at 60×, N = 7; and (F and H) HUVECs at 100×, N = 7. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.

Mentions: Values for Pearson’s correlation coefficient (PCC) were calculated from merged fluorescent microscopy images of GMVECs and HUVECs with concurrent internal FVIII and internal VWF detection (Table 2, Fig 5 and S2 Dataset). The mean PCC values (0.77–0.79) resulting from FVIII and VWF detection in HUVEC and GMVEC images indicate: a correlation in location (WPBs); and a distribution of FVIII and VWF in proportional amounts within both types of ECs (Table 2). Intensity scatter plots, shown for representative images of FVIII and VWF in WPBs of GMVECs and HUVECs, show singular linear relationships (Fig 5C, 5D, 5G and 5H).


Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

Turner NA, Moake JL - PLoS ONE (2015)

Statistical confirmation of the presence of FVIII and VWF in WPBs.GMVECs and HUVECs were treated with Triton-X to allow internal staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse IgG AF-647 (red) followed by staining with rabbit anti-human VWF plus chicken anti-rabbit IgG AF-488 (green). Merged images of FVIII and VWF detection, along with the corresponding intensity scatter plot, are shown in: (A and C) GMVECs at 60×, N = 4; (B and D) GMVECs at 100×, N = 5; (E and G) HUVECs at 60×, N = 7; and (F and H) HUVECs at 100×, N = 7. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608722&req=5

pone.0140740.g005: Statistical confirmation of the presence of FVIII and VWF in WPBs.GMVECs and HUVECs were treated with Triton-X to allow internal staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse IgG AF-647 (red) followed by staining with rabbit anti-human VWF plus chicken anti-rabbit IgG AF-488 (green). Merged images of FVIII and VWF detection, along with the corresponding intensity scatter plot, are shown in: (A and C) GMVECs at 60×, N = 4; (B and D) GMVECs at 100×, N = 5; (E and G) HUVECs at 60×, N = 7; and (F and H) HUVECs at 100×, N = 7. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.
Mentions: Values for Pearson’s correlation coefficient (PCC) were calculated from merged fluorescent microscopy images of GMVECs and HUVECs with concurrent internal FVIII and internal VWF detection (Table 2, Fig 5 and S2 Dataset). The mean PCC values (0.77–0.79) resulting from FVIII and VWF detection in HUVEC and GMVEC images indicate: a correlation in location (WPBs); and a distribution of FVIII and VWF in proportional amounts within both types of ECs (Table 2). Intensity scatter plots, shown for representative images of FVIII and VWF in WPBs of GMVECs and HUVECs, show singular linear relationships (Fig 5C, 5D, 5G and 5H).

Bottom Line: In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs.The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H.Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Rice University, Houston, Texas, United States of America.

ABSTRACT
The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

No MeSH data available.


Related in: MedlinePlus