Supplemental Material & Data
Additional Methods & Materials
Materials
RPMI-40 and DMEM media, collagenase (type 1A), hexadimethrine bromide (Polybrene), cycloheximide, histamine, amastatin, (-)-isoproterenol, forskolin, 3-isobutyl-1-methylxanthine (IBMX), phorbol-12-myristate-13-acetate (PMA) and all other chemicals unless noted were purchased from Sigma (St. Louis, MO). Ionomycin (free acid) was obtained from CalBiochem (La Jolla, CA), leukotriene C4 (LTC4) from BioMol (Plymouth Meeting, PA), RetroNectin (CH-296) from Takara (Shiga, Japan), and sodium heparin from Lyphomed (Deerfield, IL). Additionally, Endothelial Cell Growth Supplement (ECGS) from Collaborative Biomedical Research (Bedford, MA), fetal bovine serum (fbs) and donor horse serum (dhs) from HyClone (Logan, UT), normal donkey serum (NDS) from Jackson ImmunoResearch Laboratories (West Grove, PA), and Vectashield mounting medium from Vector Labs (Burlingame, CA). A synthetic thrombin-receptor activated peptide (TRAP) consisting of SFLLRNPNDKY was synthesized by the Blood Research Institute Peptide Core Laboratory.
Antibodies
Monoclonal antibodies (mAb) MBC 103.3 (anti-human FVIII), AvW-1(anti-human vWF), 1.3 (anti-human PECAM), and polyclonals (pAb) against vWF, vW AgII, and FVIII used in the immunodetection and ELISA assays were produced by the Blood Research Institute Hybridoma Laboratory and purified using Protein A columns. Other endothelial cell marker proteins were identified using anti-P-selectin (C-20) from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-Cathepsin D (Ab-2) from CalBiochem. The fluorescein (FITC)- and Texas Red (TXR)-conjugated secondary antibodies [F(ab’)2 fragment donkey anti-mouse, or anti-rabbit, or anti-goat IgG (H+L)] used in the immunofluorescent stainings were all obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Preparation of Tissues and Cell Cultures
Isolation of primary human umbilical vein endothelial cells (HUVEC) was performed by collagenase digestion of freshly obtained umbilical cord segments as previously described.1 Endothelial cells from three to five cords were isolated and pooled for each experiment, and then grown in complete primary HUVEC medium [RPMI medium supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES (9 mM), ECGS (125 mg/ml), heparin (6.45 U/ml), and 15% dhs]. Primary HUVECs were cultured on either BioCoat Collagen I precoated 6-well plates (Becton-Dickinson, Bedford, MA) or on Falcon 6-well plates coated with RetroNectin (CH-296) at optimal conditions (96 mg/ml, 20mg/cm2). Isolation of bovine aortic endothelial cells (BAEC) from a single aorta segment was previously described,2 and were used in this study as third passage cells seeded on gelatin-coated 6-well plates. Other cell lines (HT-1080, Hep-G2) were similarly cultured on 6-well plates (uncoated) in DMEM medium with 10% fbs. For the retroviral transduction experiments, these cultured media were changed to DMEM/HUVEC medium [DMEM containing 10% fbs, heparin (6.45 U/ml), and ECGS (50 mg/ml)] ninety-six hours following the initial transduction. All cell cultures were grown at 37°C in a humidified incubator with 5% CO2.
Supplemental Results
Immunolocation of FVIII and Other Endothelial Cell Antigens
Further analysis of FVIII-transduced HUVECs was performed by immunofluorescent microscopy to confirm that granules containing both vWF and FVIII were Weibel-Palade bodies and not other types of granules. First, antibodies against two specific WPB proteins (vW-AgII and P-selectin) were employed. Both vW-AgII (Figure I, panels A-C) and P-selectin (panels D-F) colocalized with FVIII in storage granules. As expected, cleaved vWF propeptide (vW-AgII) was stored in WPBs in every cell (Figure I, panels B and C). Likewise, the Weibel–Palade coat protein, P-selectin3 was observed in storage granules in endothelial cells (panels E and F). When the transduced HUVECs were dual labeled for FVIII and vW-AgII or P-selectin, there is granular stored FVIII (panels B and E) colocalized with both proteins (panels C and F). Second, additional cultures of retrovirally-transduced cells were labeled with anti-cathepsin D (lysosomes) or anti-PECAM (transmembrane protein) in combination with anti-FVIII antibodies to confirm the storage granule type. Cathepsin D is present only in smaller lysosomal vesicles,4 whereas, PECAM is present on the cell surface of endothelial cells.5 FVIII detected did not colocalize with either cathepsin D (not shown) or PECAM (Figure I, panels G-I). FVIII only was detected localized to granules displaying proteins of the WPBs.
Supplemental Figure Legends
Figure I.
Colocalization of FVIII and other endothelial cell proteins in storage granules. hFVIII(v)-transduced primary HUVECs were fixed and stained as described for Figure 1, except that different polyclonal antibodies were employed to detect other endothelial proteins. (A-C) The cells were dual labeled for FVIII and vW-AgII. (D-F) The cells were dual labeled for FVIII and P-selectin (CD62-P). (G-I) The cells were dual labeled for FVIII and PECAM (CD31). The panels demonstrate the FITC detection of FVIII (A, D, and G), TXR detection of depositions of vW-AgII, P-selectin, and PECAM, respectively (B, E, and H), and the merge of the FITC and TXR images (C, F, and I). (x2512 total enlargement). Only vW-AgII and P-selectin colocalize with FVIII in granules (yellow in merges), hence FVIII is stored in WP-bodies. PECAM detection remains localized to the cell borders.
Supplemental References
1. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745-2756.
2. Schwartz SM. Selection and characterization of bovine aortic endothelial cells. In Vitro 1978;14:966-980.
3. Bonfanti R, Furie BC, Furie B, Wagner DD. PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells. Blood 1989;73:1109-1112.
4. Contractor SF, Krakauer K. Immunofluorescent localisation of cathepsin D in trophoblastic cells in tissue culture. Beitr Pathol 1976;158:445-449.
5. Newman PJ. The biology of PECAM-1. J Clin Invest 1997;100:S25-S29.