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von Willebrand factor (vWf) serves to bridge between receptors on the platelet cytoplasmic membrane and the extracellular matrix. In addition to circulating in plasma, vWf is deposited into the extracellular matrix of the subendothelium where it is associated with type VI collagen microfibrils, but not with the elastin-associated microfibrils which are present in the deepest portion of the subendothelium at the zone of the internal elastic lamina. The reaction of platelets to type VI collagen in flow systems is qualitatively different from the shear rate dependent adhesion and aggregation response which is observed with fibrillar type I collagen, exhibiting a response only at low shear rates. The adhesion response to type VI collagen is dependent upon vWf, GP Ib and the GP IIb-IIIa complex. Platelets exposed to purified fibrillin-containing elastin-associated microfibrils adhere and aggregate at low shear rates; this response appears to involve GP IIb-IIIa but not GP Ib. The data are consistent with the hypothesis that type VI collagen is a physiologically relevant binding site for vWf in subendothelium.

von Willebrand factor (vWF) plays an important role in the process of platelet adhesion after endothelial injury by serving as a bridge between constituents of the vascular subendothelium and platelet membrane receptors. We previously presented evidence that type VI collagen microfibrils serve as a binding site for vWF in human vascular subendothelium. However, others have proposed that vWF is not associated with type VI collagen but rather with the thicker elastin-associated microfibrils, which contain several proteins including fibrillin. We therefore investigated the relationships among vWF, type VI collagen, and fibrillin in human vascular subendothelium by immunoelectron microscopy using single- and double-labeling immunogold localization techniques. In addition, we observed the three-dimensional ultrastructure of vWF-microfibril complexes by stereo paired micrographs and stereo viewer. We found that vWF co-localizes only with the type VI collagen microfibrils in subendothelium but not with fibrillin microfibrils or striated collagen. The vWF is present in subendothelium in the form of electron-dense aggregates having diameters varying between 65 and 80 nm that are closely associated with, and enmesh, the type VI collagen microfibrils and have structural similarities to intracellular Weibel-Palade bodies. The occasional co-localization of type VI collagen and fibrillin adjacent to internal elastic lamina was observed. These results are consistent with the hypothesis that type VI collagen, but not fibrillin-containing microfibrils, serves as a physiologically relevant binding site for vWF in the vascular subendothelium, where the type VI collagen-vWF complex may play an important role modulating the hemostatic response to vascular injury.

The binding of von Willebrand factor (vWF) to subendothelium constitutes an important initial step in the process of platelet adhesion to exposed subendothelium following blood vessel injury. We previously demonstrated that vWF is present in human vascular subendothelium and recently found that a 150 kd vWF-binding protein, which we extracted from subendothelium, is type VI collagen. Although we have established that vWF and type VI collagen bind in vitro, it is not known whether these two proteins are associated in the vascular subendothelium in situ. We, therefore, 1) investigated the morphological effects of our biochemical extraction procedure on human umbilical veins by scanning and transmission electron microscopy, 2) analyzed the subendothelial extract by immunofluorescence for the presence of vWF and collagens and by electron microscopy for morphological characteristics, and 3) localized vWF and type VI collagen in subendothelium by immunofluorescence and by single- and double-label immunoelectron microscopic studies with protein A-conjugated gold particles. We found that the surface exposed following de-endothelialization is composed of microfibrils and contains very little fibrillar collagen. The subendothelium is stripped after sodium dodecyl sulfate-urea extraction, and the extract itself contains immunoreactive vWF and type VI collagen but no immunoreactive type I or III fibrillar collagens. Immunofluorescence and immunoelectron microscopic studies showed that vWF and type VI collagen are both present in subendothelium, where both co-localized to microfibrils. In conclusion, vWF that binds to type VI collagen in vitro, also co-localizes with type VI collagen in subendothelium, where both are associated with microfibrils. Type VI collagen, therefore appears to serve as a biologically significant binding site for vWF in vivo and may thereby play a role in mediating platelet adhesion to exposed subendothelium following vascular injury.

Immunologic function of endothelial cells is especially important in consideration of vein allografting for arterial reconstruction and in organ allotransplantation. Ultraviolet B radiation (UVB) has previously been shown to modulate graft immunogenicity, and to alter cell surface receptor function. In this study, superficial epigastric veins were UVB irradiated with 10, 24, 40, 80, and 150 mJ/cm2 while control veins were not irradiated; all specimens were examined for endothelial ultrastructural changes. Veins were perfuse-fixed at 1, 3, 7, 14, and 28 days after irradiation, and were evaluated by transmission electron microscopy and scanning electron microscopy. Control veins had a normal appearing endothelial lining, composed of elongated, attenuated endothelial cells. Veins irradiated with more than 24 mJ/cm2 displayed injured endothelial cells characterized by altered microvilli, defects in the cell surface, and a change in cell shape. The degree of cell damage correlated closely with increasing UVB dose. At doses of 80 mJ/cm2 or greater there was moderate to severe endothelial cell separation from the underlying basement membrane and an increase in cellular lysosomes. The effects of UVB were maximal at 3 days with virtual recovery in resurfacing of all specimens with endothelium 28 days after irradiation. These data suggest that UVB has a dose-dependent effect on venous endothelium that is morphologically reversible with time. Cell membrane changes seen following exposure to UVB may contribute to altered cell surface receptor function.

Prosthetic grafts of vein allografts are inadequate as small-diameter vessel substitutes. We have applied ultraviolet B (UVB) irradiation to modulate the immunogenicity of vein allografts to avoid immunologic injury. The veins of male ACI rats were irradiated with UVB (60 mJ/cm2) in situ and transplanted to male ACI rats (autografts) and female Lewis rats (allografts). Nonirradiated veins served as controls. At 4, 7, 14, and 28 days, all grafts were patent and were studied for morphologic changes by scanning electron microscopy and for immunogold labeling of major histocompatibility complex class II antigen expression. In autografts, scanning electron microscopy demonstrated minimal endothelial loss after grafting, regardless of UVB irradiation. Untreated allografts showed severe endothelial injury 4, 7, and 14 days after transplantation. UVB irradiation of veins protected allografts from injury to the endothelium and basement membrane. Major histocompatibility complex class II-positive endothelial cells were not seen in autografts but were seen in 40% of cells 4 days after transplantation in untreated allografts. UVB-treated allografts showed MHC class II antigen expression labeling of 20% of the endothelial cells. Barr body analysis demonstrated the donor origin of these endothelial cells. UVB irradiation of rat vein allografts prolongs endothelial survival while decreasing endothelial surface expression of class II antigens. These data suggest that modification of vein immunogenicity with UVB irradiation may permit functional survival of small-vessel allografts without chronic immunosuppression.

MHC class II antigens (Ia) are important to the primary transplantation response. The present study investigates the kinetics of expression of Ia antigens on endothelial cells of rat vein autografts and allografts. The superficial epigastric veins of 15 ACI rats were anastomosed to the femoral artery of Lewis recipients using standard microsurgical technique. Eighteen Lewis-to-Lewis or ACI-to-ACI autografts served as controls. Veins were studied for Ia expression at 1, 4, and 7 days after grafting using the immunogold electron microscopic technique. All grafts remained patent for the duration of the experiment. Endothelial cells from control grafts did not demonstrate labeling of Ia antigens on their cell surfaces. Similarly, few mononuclear-adventitial cells in control grafts were Ia positive. When endothelial cells from vein allografts were studies for Ia antigen expression, 40% of the endothelial cells showed labeling for Ia at 4 days after transplantation. At 1 week following grafting, endothelial survival was inadequate for study because of cell loss secondary to rejection. In comparison to autografts, rejecting vein allografts showed numerous Ia-positive mononuclear cells permeating the vessel adventitia. Expression of Ia antigens by vascular endothelium may enable these cells to perform tasks traditionally ascribed to monocytes or dendritic cells. Effort to alter Ia expression may suggest an approach to long-term vein allograft survival.