Acetylated-low density lipoprotein (Ac-LDL) is taken up by macrophages and endothelial cells via the "scavenger cell pathway" of LDL metabolism. In this report, aortic and microvascular endothelial cells internalized and degraded 7-15 times more [1251]-Ac-LDL than did smooth muscle cells or pericytes. Bound [12Sl]-Ac-LDL was displaced by unlabeled Ac-LDL, but not unmodified LDL. The ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL labeled with the fluorescent probe 1,1 '-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). When cells were incubated with 10 pg/ml DiI-Ac-LDL for 4 h at 37°C and subsequently examined by fluorescence microscopy, capillary and aortic endothelial cells were brilliantly fluorescent whereas the fluorescent intensity of retinal pericytes and smooth muscle cells was only slightly above background levels. DiI-Ac-LDL at the concentration used for labeling cells had no effect on endothelial cell growth rate. When primary cultures of bovine adrenal capillary cells were labeled with 10 pg/ml of DiI-Ac-LDL for 4 h at 37°C, then trypsinized and subjected to fluorescence-activated cell sorting, pure cultures of capillary endothelial cells could be obtained. Utilizing this method, large numbers of early passage microvascular endothelial cells can be obtained in significantly less time than with conventional methods.A major problem in the study of microvascular endothelial cells is the identification of the desired cell population and the subsequent isolation of pure cultures. Our currently used method of establishing pure cultures of capillary endothelial cells involves many weeks of "weeding out" nonendothelial cells (l). The weeding technique involves the assumption that the morphology of capillary endothelial cells is similar to other endothelial cells and is thus directed at isolating colonies with those characteristics. Several markers for endothelial cells are routinely used for confirmation that established cell lines are of endothelial origin. These include the presence of factor VIII related antigen (2, 3) and angiotensin converting enzyme (4, 5). Microvascular endothelial cells differ from large vessel endothelial cells in their requirement for additional growth factors and modified surfaces for optimal growth (l), and their response to tumor factors (l, 6).The receptor-mediated uptake of low density lipoprotein (LDL ~) by cells has been studied in detail (for a review see reference 7). An alternative pathway for the metabolism of chemically modified lipoproteins has also been described (8) and has been termed the "scavenger cell pathway" of LDL metabolism, due to its occurrence in rodent and canine macrophages (9-11) and human monocytes (12). Various chemical methods for modification of LDL have been used to modify the charge of amino groups on LDL including acetylation (8), acetoacetylation (9), and malondialdehyde treatment (12). These modified lipoproteins are taken up by ~Abbreviations us...

We have synthesized and studied two 1,2-dioxetane-based chemiluminescent enzyme substrates: 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD), and, 3-(2'-spiroadamantane)-4-methoxy-4-(3"-beta-D'-galactopyrano -yloxy)phenyl-1,2- dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and beta-D-galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which 'stabilizes' the dephosphorylated AMPPD emitter. Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer. AMPPD and AMPGD offer alternatives to colorimetric and fluorescent substrates for alkaline phosphatase and beta-D-galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. beta-hCG, LH, TSH and others).

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