Figure 2
Figure 2. Subcellular distribution and expression of VWF in the BOECs isolated from the IP and healthy donors. (A) Characteristics of the BOECs via staining of the cell-specific markers VWF (light green) and PECAM (red) with secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-594, respectively. However, only 64 of 100 inspected IP BOECs emitted light green fluorescent signals, representing production of VWF protein. In the VWF-expressing IP BOECs, VWF staining is mostly diffuse, accumulating around the nucleus of the cells, whereas in normal BOECs, VWF can be seen as distinct elongated structures, indicating storage in WPBs. The BOECs isolated from the IP’s mother illustrate a combination of VWF stored in WPBs and diffuse staining. The white box points out secreting VWF strings in normal BOECs that are not visible in the IP-derived BOECs. Bars represent 20 µm. (B) Diffuse staining observed in BOECs obtained from the IP and her mother (carrying the mutation c. 7464C>T) was colocalized with protein disulfide isomerase (PDI). Staining was performed using primary antibodies anti-VWF (left channel, light green) and anti-PDI (ER marker; middle, red) and secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-555, respectively. Colocalization of VWF and PDI staining is illustrated in the right channel (Merge). Bars represent 10 µm. (C) Bar graph of the mean of VWF:Ag levels in the medium and lysates of BOECs obtained from the IP, her mother (3 independent experiments, N = 3), and 3 healthy donors (each 3 independent experiments, N = 9). The mean VWF:Ag was determined after 10× concentration of the collected medium and cell lysates of confluent BOECs in 75 cm2 flasks. Error bars indicate the standard deviation. (D) Western blot analysis of BOEC intracellular VWF after electrophoresis on 4% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Left lane shows both pre-pro-VWF and mature VWF in the lysate of the normal BOECs after 10× concentration. Middle and right lanes are representative of the IP’s BOEC lysates after 10× and 30× concentrations, respectively. WPBs, Weibel-Palade bodies.

Subcellular distribution and expression of VWF in the BOECs isolated from the IP and healthy donors. (A) Characteristics of the BOECs via staining of the cell-specific markers VWF (light green) and PECAM (red) with secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-594, respectively. However, only 64 of 100 inspected IP BOECs emitted light green fluorescent signals, representing production of VWF protein. In the VWF-expressing IP BOECs, VWF staining is mostly diffuse, accumulating around the nucleus of the cells, whereas in normal BOECs, VWF can be seen as distinct elongated structures, indicating storage in WPBs. The BOECs isolated from the IP’s mother illustrate a combination of VWF stored in WPBs and diffuse staining. The white box points out secreting VWF strings in normal BOECs that are not visible in the IP-derived BOECs. Bars represent 20 µm. (B) Diffuse staining observed in BOECs obtained from the IP and her mother (carrying the mutation c. 7464C>T) was colocalized with protein disulfide isomerase (PDI). Staining was performed using primary antibodies anti-VWF (left channel, light green) and anti-PDI (ER marker; middle, red) and secondary antibodies conjugated with Alexa Fluor-488 and Alexa Fluor-555, respectively. Colocalization of VWF and PDI staining is illustrated in the right channel (Merge). Bars represent 10 µm. (C) Bar graph of the mean of VWF:Ag levels in the medium and lysates of BOECs obtained from the IP, her mother (3 independent experiments, N = 3), and 3 healthy donors (each 3 independent experiments, N = 9). The mean VWF:Ag was determined after 10× concentration of the collected medium and cell lysates of confluent BOECs in 75 cm2 flasks. Error bars indicate the standard deviation. (D) Western blot analysis of BOEC intracellular VWF after electrophoresis on 4% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Left lane shows both pre-pro-VWF and mature VWF in the lysate of the normal BOECs after 10× concentration. Middle and right lanes are representative of the IP’s BOEC lysates after 10× and 30× concentrations, respectively. WPBs, Weibel-Palade bodies.

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