A procedure for the isolation and in vitro cultivation of endothelial cells from the microvessels of the newborn human foreskin dermis is described. The epidermis was removed from foreskin tissue using a Castroviejo keratotome (0.1 mm shim). Endothelial cells were released from the dermal vessels by trypsinization of 5 mm2 sections of dermis at 37 degrees C for 40 min. Cells were expressed into Minimal Essential Medium (MEM) containing 10% pooled human serum, collected by centrifugation and plated onto either a plain plastic or a fibronectin treated culture surface. In primary culture the rate of endothelial cell proliferation was dependent upon serum type and concentration being optimal in 50% pooled human serum. High serum concentration in combination with pretreatment of the culture surface with fibronectin was required for maximal proliferation rate, for the cells to achieve confluence and for subcultivation. Primary and subcultured cells were characterized as endothelial by light microscopic, immunofluorescent (Factor VIII associated protein) and ultrastructural (Weibel-Palade body) criteria.

Experiments were carried out in vitro and in normal human subjects to evaluate alternative food-grade viscous polysaccharides as agents for reducing postprandial hyperglycemia and to assess the relationship between the in vitro and in vivo performance of the polysaccharides. A 1:1 mixture of xanthan and locust bean gum (X/LBG) had the greatest viscosity at equivalent concentrations and shear rates and was more effective than guar gum, xanthan, or locust-bean gum at inhibiting glucose movement in vitro. It was not, however, more efficient in lowering postprandial blood glucose and plasma insulin in human subjects when incorporated in a drink containing 50 g glucose. When the different gums were acidified and reneutralized to mimic conditions in the gut, there was a better correlation between viscosity and blood glucose and plasma insulin levels. This effect may explain why X/LBG was no more effective than the other gums in reducing postprandial hyperglycemia in man.

Aatract. Prostaglandin I2 (PGI2), a potent vasodilator and inhibitor of platelet aggregation, is a major product of arachidonic acid metabolism in endothelial cells that are derived from large blood vessels (e.g., umbilical veins). We have examined whether PGI2 is also a major product of arachidonic acid metabolism in cultured endothelial cells that are derived from dermal microvessels in human newborn foreskin. Supernatants from confluent monolayers of endothelial cells that had been incubated for 20 min with [3H]arachidonic acid and the calcium ionophore A23187 (10 uM) were assayed for prostaglandin F2a (PGF2a), prostaglandin E2 (PGE2), and 6-keto-prostaglandin Fl, (PGFIa) (the stable metabolite of PGI2) by using authentic standards and high performance liquid chromatography. Whereas supernates from stimulated umbilical vein endothelial cells contained 6-keto-PGFia > PGF2a. > PGE2, supernates from stimulated foreskin microvessel endothelial cells contained PGF2a _ PGE2 > 6-keto-PGFIa. Similar results were obtained when supernates from stimulated, unlabeled endothelial cells were analyzed by radioimmunoassay. These data indicate that PGI2 is not a major metabolite of arachidonic acid in cultured endothelial cells from human foreskin microvessels.Portions of this work were presented at

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined . MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous . Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix . When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina raraand lamina densa-like regions . The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.Capillary endothelium lie on a basal lamina that has a number of important functions. Not only does it provide the substratum for endothelial cell attachment and tissue organization (52), but it also has a role in blood vessel permeability (15), in the initiation of blood clotting (2), and provides a barrier to cellular migration, the violation of which has importance for angiogenesis (17), leukocyte emigration (55), and tumor metastasis (35,38). Alterations in vascular basal lamina have been implicated in a number of pathological states, including diabetes mellitus, thrombogenesis, atherosclerosis, and renal failure (3,15,28,53).The vascular basal lamina is a complex structure . Because of difficulties in isolating sufficient quantities of purified material, biochemical studies of its molecular composition and structure have been limited . It is known, however, that vascular basal lamina is composed of a number of macromolecules including type IV collagen, proteoglycans, and glycoproteins (reviewed in 32, 37, 50). Studies on the synthesis of basal lamina macromolecules and their assembly into mature basal lamina by endothelia has been even more difficult. Recent advances in the isolation and long term culture of microvascular endothelial cells (3,10,17,40) provides the opportunity for the study of these events. 692

Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitro. In the presence of 5 X 10(-4) M dibutyryl cyclic AMP (Bt2cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of controls values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt2cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt2cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methylxanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40-fold. Human umbilical vein cells in culture failed to respond to either Bt2cAMP or CT together with IMX. The growth-promoting effect of dibutyryl cyclic AMP (Bt2cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.