Limits...
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

FVIII and VWF are present in the WPBs of GMVECs and HUVECs.Unstimulated GMVECs and HUVECs were fixed and treated with Triton-X to allow intracellular staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse AF IgG-647 (red), followed by staining with rabbit anti-human VWF plus chicken anti-rabbit AF IgG-488 (green). GMVEC images: (A) anti-FVIII detection (red); (B) anti-VWF detection (green); and (C) merged image of anti-FVIII plus anti-VWF. HUVEC images: (D) anti-FVIII detection (red); (E) anti-VWF detection (green); and (F) anti-FVIII plus anti-VWF. Images at 60× are shown merged with DAPI-detected nuclei (gray) and are representative of 5–7 experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608722&req=5

pone.0140740.g003: FVIII and VWF are present in the WPBs of GMVECs and HUVECs.Unstimulated GMVECs and HUVECs were fixed and treated with Triton-X to allow intracellular staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse AF IgG-647 (red), followed by staining with rabbit anti-human VWF plus chicken anti-rabbit AF IgG-488 (green). GMVEC images: (A) anti-FVIII detection (red); (B) anti-VWF detection (green); and (C) merged image of anti-FVIII plus anti-VWF. HUVEC images: (D) anti-FVIII detection (red); (E) anti-VWF detection (green); and (F) anti-FVIII plus anti-VWF. Images at 60× are shown merged with DAPI-detected nuclei (gray) and are representative of 5–7 experiments.

Mentions: Internal staining of unstimulated GMVECs and HUVECs, using target-specific antibodies (Fig 1), demonstrates the presence of FVIII and VWF in WPBs (Fig 3 and S1 Fig). FVIII and VWF were distinctly identified by using mono-specific primary antibodies combined with secondary detection antibodies with widely separated wavelengths for excitation and emission, as shown in Fig 2. [24,26] GMVECs and HUVECs stained with secondary detection antibodies alone showed only background fluorescence (S2 Fig).


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)

FVIII and VWF are present in the WPBs of GMVECs and HUVECs.Unstimulated GMVECs and HUVECs were fixed and treated with Triton-X to allow intracellular staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse AF IgG-647 (red), followed by staining with rabbit anti-human VWF plus chicken anti-rabbit AF IgG-488 (green). GMVEC images: (A) anti-FVIII detection (red); (B) anti-VWF detection (green); and (C) merged image of anti-FVIII plus anti-VWF. HUVEC images: (D) anti-FVIII detection (red); (E) anti-VWF detection (green); and (F) anti-FVIII plus anti-VWF. Images at 60× are shown merged with DAPI-detected nuclei (gray) and are representative of 5–7 experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140740.g003: FVIII and VWF are present in the WPBs of GMVECs and HUVECs.Unstimulated GMVECs and HUVECs were fixed and treated with Triton-X to allow intracellular staining. Cells were then stained with mouse monoclonal anti-human FVIII plus goat anti-mouse AF IgG-647 (red), followed by staining with rabbit anti-human VWF plus chicken anti-rabbit AF IgG-488 (green). GMVEC images: (A) anti-FVIII detection (red); (B) anti-VWF detection (green); and (C) merged image of anti-FVIII plus anti-VWF. HUVEC images: (D) anti-FVIII detection (red); (E) anti-VWF detection (green); and (F) anti-FVIII plus anti-VWF. Images at 60× are shown merged with DAPI-detected nuclei (gray) and are representative of 5–7 experiments.
Mentions: Internal staining of unstimulated GMVECs and HUVECs, using target-specific antibodies (Fig 1), demonstrates the presence of FVIII and VWF in WPBs (Fig 3 and S1 Fig). FVIII and VWF were distinctly identified by using mono-specific primary antibodies combined with secondary detection antibodies with widely separated wavelengths for excitation and emission, as shown in Fig 2. [24,26] GMVECs and HUVECs stained with secondary detection antibodies alone showed only background fluorescence (S2 Fig).

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