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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

Fluorescent emission “cross-talk” controls.The high concentration of VWF present in Weibel-Palade bodies (WPBs) was used to demonstrate that images detected at the first wavelength channel (488 nm) were not cross contaminated by fluorescence generated from the second wavelength channel (647 nm), and reciprocally, the 647 nm channel was not affected by fluorescence from the 488 nm channel. Un-stimulated HUVECs were fixed with p-formaldehyde and treated with Triton-X to allow intracellular fluorescent staining. Cell nuclei were stained with DAPI (blue) and cells were imaged with a 60× objective. In (A and B) HUVECs on coverslip 1 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG Alexa Fluor (AF)-488 (green) and in (C and D) HUVECs on coverslip 2 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). Both coverslips were imaged at 488 nm and at 647 nm and merged with DAPI images. The images in (A) and (C) were detected at 488 nm and the images in (B) and (D) were detected at 647 nm.
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pone.0140740.g002: Fluorescent emission “cross-talk” controls.The high concentration of VWF present in Weibel-Palade bodies (WPBs) was used to demonstrate that images detected at the first wavelength channel (488 nm) were not cross contaminated by fluorescence generated from the second wavelength channel (647 nm), and reciprocally, the 647 nm channel was not affected by fluorescence from the 488 nm channel. Un-stimulated HUVECs were fixed with p-formaldehyde and treated with Triton-X to allow intracellular fluorescent staining. Cell nuclei were stained with DAPI (blue) and cells were imaged with a 60× objective. In (A and B) HUVECs on coverslip 1 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG Alexa Fluor (AF)-488 (green) and in (C and D) HUVECs on coverslip 2 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). Both coverslips were imaged at 488 nm and at 647 nm and merged with DAPI images. The images in (A) and (C) were detected at 488 nm and the images in (B) and (D) were detected at 647 nm.

Mentions: The experiments shown in Fig 2 were conducted to verify the absence of fluorescent “cross-talk” or “bleed-through” between the channels used to detect and distinguish the proteins in this study by fluorescent microscopy: 488 nm (green) and 647 nm (red). Images of HUVECs internally stained only with rabbit anti-VWF + chicken anti-rabbit IgG AF-488 (green, Fig 2A) acquired using 647-channel filters did not show detectable 647 fluorescence (Fig 2B). HUVECs stained only with rabbit anti-VWF + chicken anti-rabbit IgG AF-647 (red, Fig 2D) acquired using 488-channel filters did not show any 488 fluorescence signal (Fig 2C).


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)

Fluorescent emission “cross-talk” controls.The high concentration of VWF present in Weibel-Palade bodies (WPBs) was used to demonstrate that images detected at the first wavelength channel (488 nm) were not cross contaminated by fluorescence generated from the second wavelength channel (647 nm), and reciprocally, the 647 nm channel was not affected by fluorescence from the 488 nm channel. Un-stimulated HUVECs were fixed with p-formaldehyde and treated with Triton-X to allow intracellular fluorescent staining. Cell nuclei were stained with DAPI (blue) and cells were imaged with a 60× objective. In (A and B) HUVECs on coverslip 1 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG Alexa Fluor (AF)-488 (green) and in (C and D) HUVECs on coverslip 2 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). Both coverslips were imaged at 488 nm and at 647 nm and merged with DAPI images. The images in (A) and (C) were detected at 488 nm and the images in (B) and (D) were detected at 647 nm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140740.g002: Fluorescent emission “cross-talk” controls.The high concentration of VWF present in Weibel-Palade bodies (WPBs) was used to demonstrate that images detected at the first wavelength channel (488 nm) were not cross contaminated by fluorescence generated from the second wavelength channel (647 nm), and reciprocally, the 647 nm channel was not affected by fluorescence from the 488 nm channel. Un-stimulated HUVECs were fixed with p-formaldehyde and treated with Triton-X to allow intracellular fluorescent staining. Cell nuclei were stained with DAPI (blue) and cells were imaged with a 60× objective. In (A and B) HUVECs on coverslip 1 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG Alexa Fluor (AF)-488 (green) and in (C and D) HUVECs on coverslip 2 were stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). Both coverslips were imaged at 488 nm and at 647 nm and merged with DAPI images. The images in (A) and (C) were detected at 488 nm and the images in (B) and (D) were detected at 647 nm.
Mentions: The experiments shown in Fig 2 were conducted to verify the absence of fluorescent “cross-talk” or “bleed-through” between the channels used to detect and distinguish the proteins in this study by fluorescent microscopy: 488 nm (green) and 647 nm (red). Images of HUVECs internally stained only with rabbit anti-VWF + chicken anti-rabbit IgG AF-488 (green, Fig 2A) acquired using 647-channel filters did not show detectable 647 fluorescence (Fig 2B). HUVECs stained only with rabbit anti-VWF + chicken anti-rabbit IgG AF-647 (red, Fig 2D) acquired using 488-channel filters did not show any 488 fluorescence signal (Fig 2C).

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