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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 images of stained fibroblasts.Fibroblasts were stained with antibodies to Fibroblast Surface Protein (FSP), FVIII, and VWF plus relevant secondary antibodies, or with secondary detection antibodies alone. Images at 60× from each channel plus the merged image are shown. Cell nuclei were detected with DAPI (blue). (A-C) Fibroblasts were fixed and cell surfaces were stained with mouse anti-FSP plus goat anti-mouse IgM AF-488 (green). Cells were fixed again (to retain surface antibodies), and then treated with Triton-X for internal staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). (D-F) Fibroblasts were fixed and treated with Triton-X for internal staining. Cells were stained with mouse anti-FVIII plus goat anti-mouse IgG AF-647 (red), followed by staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green). (G-I) Fixed and Triton-X-treated Fibroblasts were stained with only the secondary fluorescently labeled antibodies used in panels D-F for FVIII and VWF detection. Cells were stained with goat anti-mouse IgG AF-647 (red), and then with chicken anti-rabbit IgG AF-488 (green). Images are representative of 3 experiments.
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pone.0140740.g004: Fluorescent images of stained fibroblasts.Fibroblasts were stained with antibodies to Fibroblast Surface Protein (FSP), FVIII, and VWF plus relevant secondary antibodies, or with secondary detection antibodies alone. Images at 60× from each channel plus the merged image are shown. Cell nuclei were detected with DAPI (blue). (A-C) Fibroblasts were fixed and cell surfaces were stained with mouse anti-FSP plus goat anti-mouse IgM AF-488 (green). Cells were fixed again (to retain surface antibodies), and then treated with Triton-X for internal staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). (D-F) Fibroblasts were fixed and treated with Triton-X for internal staining. Cells were stained with mouse anti-FVIII plus goat anti-mouse IgG AF-647 (red), followed by staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green). (G-I) Fixed and Triton-X-treated Fibroblasts were stained with only the secondary fluorescently labeled antibodies used in panels D-F for FVIII and VWF detection. Cells were stained with goat anti-mouse IgG AF-647 (red), and then with chicken anti-rabbit IgG AF-488 (green). Images are representative of 3 experiments.

Mentions: In contrast to GMVECs and HUVECs, FVIII and VWF proteins were not detected by fluorescent microscopy in fibroblasts (Fig 4A–4F). The fibroblasts appeared to stain positively for VWF when the chicken anti-rabbit IgG-488 was used as the secondary detection antibody (Fig 4E and 4F), and negatively for VWF with chicken anti-rabbit IgG-647 as the secondary detection antibody (Fig 4B). Intensity measurements of full image areas of the fibroblasts were made after cell staining using each secondary detection antibody and in the presence or absence of primary antibodies to VWF. The intensity measurements showed that images of fibroblasts incubated with secondary IgG-488 antibodies alone had almost 2-fold higher fluorescent intensity than images of fibroblasts detected with the primary antibody to VWF plus secondary IgG-488 antibodies (S5 Table and S7 Dataset). Fibroblasts stained with secondary mouse antibodies alone also showed equal or higher amounts of fluorescent staining than fibroblasts stained with the primary mouse anti-FVIII plus these secondary detection antibodies (Fig 4D and 4G).


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 images of stained fibroblasts.Fibroblasts were stained with antibodies to Fibroblast Surface Protein (FSP), FVIII, and VWF plus relevant secondary antibodies, or with secondary detection antibodies alone. Images at 60× from each channel plus the merged image are shown. Cell nuclei were detected with DAPI (blue). (A-C) Fibroblasts were fixed and cell surfaces were stained with mouse anti-FSP plus goat anti-mouse IgM AF-488 (green). Cells were fixed again (to retain surface antibodies), and then treated with Triton-X for internal staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). (D-F) Fibroblasts were fixed and treated with Triton-X for internal staining. Cells were stained with mouse anti-FVIII plus goat anti-mouse IgG AF-647 (red), followed by staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green). (G-I) Fixed and Triton-X-treated Fibroblasts were stained with only the secondary fluorescently labeled antibodies used in panels D-F for FVIII and VWF detection. Cells were stained with goat anti-mouse IgG AF-647 (red), and then with chicken anti-rabbit IgG AF-488 (green). Images are representative of 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140740.g004: Fluorescent images of stained fibroblasts.Fibroblasts were stained with antibodies to Fibroblast Surface Protein (FSP), FVIII, and VWF plus relevant secondary antibodies, or with secondary detection antibodies alone. Images at 60× from each channel plus the merged image are shown. Cell nuclei were detected with DAPI (blue). (A-C) Fibroblasts were fixed and cell surfaces were stained with mouse anti-FSP plus goat anti-mouse IgM AF-488 (green). Cells were fixed again (to retain surface antibodies), and then treated with Triton-X for internal staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-647 (red). (D-F) Fibroblasts were fixed and treated with Triton-X for internal staining. Cells were stained with mouse anti-FVIII plus goat anti-mouse IgG AF-647 (red), followed by staining with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green). (G-I) Fixed and Triton-X-treated Fibroblasts were stained with only the secondary fluorescently labeled antibodies used in panels D-F for FVIII and VWF detection. Cells were stained with goat anti-mouse IgG AF-647 (red), and then with chicken anti-rabbit IgG AF-488 (green). Images are representative of 3 experiments.
Mentions: In contrast to GMVECs and HUVECs, FVIII and VWF proteins were not detected by fluorescent microscopy in fibroblasts (Fig 4A–4F). The fibroblasts appeared to stain positively for VWF when the chicken anti-rabbit IgG-488 was used as the secondary detection antibody (Fig 4E and 4F), and negatively for VWF with chicken anti-rabbit IgG-647 as the secondary detection antibody (Fig 4B). Intensity measurements of full image areas of the fibroblasts were made after cell staining using each secondary detection antibody and in the presence or absence of primary antibodies to VWF. The intensity measurements showed that images of fibroblasts incubated with secondary IgG-488 antibodies alone had almost 2-fold higher fluorescent intensity than images of fibroblasts detected with the primary antibody to VWF plus secondary IgG-488 antibodies (S5 Table and S7 Dataset). Fibroblasts stained with secondary mouse antibodies alone also showed equal or higher amounts of fluorescent staining than fibroblasts stained with the primary mouse anti-FVIII plus these secondary detection antibodies (Fig 4D and 4G).

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