Macrophage-specific expression of human lipoprotein lipase accelerates atherosclerosis in transgenic apolipoprotein e knockout mice but not in C57BL/6 mice.

Transgenic mice with macrophage-specific expression of human (hu) lipoprotein lipase (LPL) were generated to determine the contribution of macrophage LPL to atherogenesis. Macrophage specificity was accomplished with the scavenger receptor A promoter. Complete characterization demonstrated that macrophages from these mice expressed huLPL mRNA and secreted enzymatically active huLPL protein. Expression of huLPL was macrophage specific, because total RNA isolated from heart, thymus, lung, liver, muscle, and adipose tissues was devoid of huLPL mRNA. Macrophage-specific expression of huLPL did not exacerbate lesions in aortas of C57BL/6 mice even after 32 weeks on an atherosclerotic diet. However, when expressed in apolipoprotein E knockout background, the extent of occlusion in the aortic sinus region of male huLPL+ mice increased 51% (n=9 to 11, P<0.002) compared with huLPL- mice after they had been fed a Western diet for 8 weeks. The proatherogenic effect of macrophage LPL was confirmed in serial sections of the aorta obtained after mice had been fed a Western diet for 3 weeks. By immunohistochemical analysis, huLPL protein was detected in the lesions of huLPL+ mice but not in huLPL- mice. Our results establish that macrophage LPL accelerates atherosclerosis in male apolipoprotein E knockout mice.

Lipoprotein lipase- and hepatic triglyceride lipase- promoted very low density lipoprotein degradation proceeds via an apolipoprotein E-dependent mechanism.

Apolipoprotein E (apoE) is the primary recognition signal on triglyceride-rich lipoproteins responsible for interacting with low density lipoprotein (LDL) receptors and LDL receptor-related protein (LRP). It has been shown that lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) promote receptor-mediated uptake and degradation of very low density lipoproteins (VLDL) and remnant particles, possibly by directly binding to lipoprotein receptors. In this study we have investigated the requirement for apoE in lipase-stimulated VLDL degradation. We compared binding and degradation of normal and apoE-depleted human VLDL and apoE knockout mouse VLDL in human foreskin fibroblasts. Surface binding at 37 degrees C of apoE knockout VLDL was greater than that of normal VLDL by 3- and 40-fold, respectively, in the presence of LPL and HTGL. In spite of the greater stimulation of surface binding, lipase-stimulated degradation of apoE knockout mouse VLDL was significantly lower than that of normal VLDL (30, 30, and 80%, respectively, for control, LPL, and HTGL treatments). In the presence of LPL and HTGL, surface binding of apoE-depleted human VLDL was, respectively, 40 and 200% of normal VLDL whereas degradation was, respectively, 25 and 50% of normal VLDL. LPL and HTGL stimulated degradation of normal VLDL in a dose-dependent manner and by a LDL receptor-mediated pathway. Maximum stimulation (4-fold) was seen in the presence LPL (1 microgram/ml) or HTGL (3 microgram/ml) in lovastatin-treated cells. On the other hand, degradation of apoE-depleted VLDL was not significantly increased by the presence of lipases even in lovastatin-treated cells. Surface binding of apoE-depleted VLDL to metabolically inactive cells at 4 degrees C was higher in control and HTGL-treated cells, but unchanged in the presence of LPL. Degradation of prebound apoE-depleted VLDL was only 35% as efficient as that of normal VLDL. Surface binding of apoE knockout or apoE-depleted VLDL was to heparin sulfate proteoglycans because it was completely abolished by heparinase treatment. However, apoE appears to be a primary determinant for receptor-mediated VLDL degradation. Our studies suggest that overexpression of LPL or HTGL may not protect against lipoprotein accumulation seen in apoE deficiency.

Hepatic triglyceride lipase promotes low density lipoprotein receptor-mediated catabolism of very low density lipoproteins in vitro.

We demonstrate here that hepatic triglyceride lipase (HTGL) enhances VLDL degradation in cultured cells by a LDL receptor-mediated mechanism. VLDL binding at 4 degrees C and degradation at 37 degrees C by normal fibroblasts was stimulated by HTGL in a dose-dependent manner. A maximum increase of up to 7-fold was seen at 10 microg/ml HTGL. Both VLDL binding and degradation were significantly increased (4-fold) when LDL receptors were up-regulated by treatment with lovastatin. HTGL also stimulated VLDL degradation by LDL receptor-deficient FH fibroblasts but the level of maximal degradation was 40-fold lower than in lovastatin-treated normal fibroblasts. A prominent role for LDL receptors was confirmed by demonstration of similar HTGL-promoted VLDL degradation by normal and LRP-deficient murine embryonic fibroblasts. HTGL enhanced binding and internalization of apoprotein-free triglyceride emulsions, however, this was LDL receptor-independent. HTGL-stimulated binding and internalization of apoprotein-free emulsions was totally abolished by heparinase indicating that it was mediated by HSPG. In a cell-free assay HTGL competitively inhibited the binding of VLDL to immobilized LDL receptors at 4 degrees C suggesting that it may directly bind to LDL receptors but may not bind VLDL particles at the same time. We conclude that the ability of HTGL to enhance VLDL degradation is due to its ability to concentrate lipoprotein particles on HSPG sites on the cell surface leading to LDL receptor-mediated endocytosis and degradation.

Lipoprotein lipase binds to low density lipoprotein receptors and induces receptor-mediated catabolism of very low density lipoproteins in vitro.

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of plasma triglycerides, promotes binding and catabolism of triglyceride-rich lipoproteins by various cultured cells. Recent studies demonstrate that LPL binds to three members of the low density lipoprotein (LDL) receptor family, including the LDL receptor-related protein (LRP), GP330/LRP-2, and very low density lipoprotein (VLDL) receptors and induces receptor-mediated lipoprotein catabolism. We show here that LDL receptors also bind LPL and mediate LPL-dependent catabolism of large VLDL with Sf 100-400. Up-regulation of LDL receptors by lovastatin treatment of normal human foreskin fibroblasts (FSF cells) resulted in an increase in LPL-induced VLDL binding and catabolism to a level that was 10-15-fold greater than in LDL receptor-negative fibroblasts, despite similar LRP activity in both cell lines. This indicates that the contribution of LRP to LPL-dependent degradation of VLDL is small when LDL receptors are maximally up-regulated. Furthermore studies in LRP-deficient murine embryonic fibroblasts showed that the level of LPL-dependent degradation of VLDL was similar to that in normal murine embryonic fibroblasts. LPL also promoted the internalization of protein-free triglyceride emulsions; lovastatin-treatment resulted in 2-fold higher uptake in FSF cells, indicating that LPL itself could bind to LDL receptors. However, the lower induction of emulsion catabolism as compared with native VLDL suggests that LPL-induced catabolism via LDL receptors is only partially dependent on receptor binding by LPL and instead is primarily due to activation of apolipoproteins such as apoE. A fusion protein between glutathione S-transferase and the catalytically inactive carboxyl-terminal domain of LPL (GST-LPLC) also induced binding and catabolism of VLDL. However GST-LPLC was not as active as native LPL, indicating that lipolysis is required for a maximal LPL effect. Mutations of critical tryptophan residues in GST-LPLC that abolished binding to VLDL converted the protein to an inhibitor of lipoprotein binding to LDL receptors. In solid-phase assays using immobilized receptors, LDL receptors bound to LPL in a dose-dependent manner. Both LPL and GST-LPLC promoted binding of VLDL to LDL receptor-coated wells. These results indicate that LPL binds to LDL receptors and suggest that the carboxyl-terminal domain of LPL contributes to this interaction.

The 39-kDa receptor-associated protein modulates lipoprotein catabolism by binding to LDL receptors.

The 39-kDa receptor-associated protein (RAP) is cosynthesized and co-purifies with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and is thought to modulate ligand binding to LRP. In addition to binding LRP, RAP binds two other members of the low density lipoprotein (LDL) receptor family, gp330 and very low density lipoprotein (VLDL) receptors. Here, we show that RAP binds to LDL receptors as well. In normal human foreskin fibroblasts, RAP inhibited LDL receptor-mediated binding and catabolism of LDL and VLDL with Sf 20-60 or 100-400. RAP inhibited 125I-labeled LDL and Sf 100-400 lipoprotein binding at 4 degrees C with KI values of 60 and 45 nM, respectively. The effective concentrations for 50% inhibition (EC50) of cellular degradation of 2.0 nM 125I-labeled LDL, 4.7 nM 125I-labeled Sf 20-60, and 3.6 nM 125I-labeled Sf 100-400 particles were 40, 70, and 51 nM, respectively. Treatment of cells with lovastatin to induce LDL receptors increased cellular binding, internalization, and degradation of RAP by 2.3-, 1.7-, and 2.6-fold, respectively. In solid-phase assays, RAP bound to partially purified LDL receptors in a dose-dependent manner. The dissociation constant (KD) of RAP binding to LDL receptors in the solid-phase assay was 250 nM, which is higher than that for LRP, gp330, or VLDL receptors in similar assays by a factor of 14 to 350. Also, RAP inhibited 125I-labeled LDL and Sf 100-400 VLDL binding to LDL receptors in solid-phase assays with KI values of 140 and 130 nM, respectively. Because LDL bind via apolipoprotein (apo) B100 whereas VLDL bind via apoE, our results show that RAP inhibits LDL receptor interactions with both apoB100 and apoE. These studies establish that RAP is capable of binding to LDL receptors and modulating cellular catabolism of LDL and VLDL by this pathway.

Cellular catabolism of normal very low density lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is induced by the C-terminal domain of lipoprotein lipase.

Lipoprotein lipase (LPL) binds to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor and induces catabolism of normal human very low density lipoproteins (VLDL) via LRP in vitro. Recent studies showed that the C-terminal domain of LPL can bind LRP in solid phase assays and inhibit cellular catabolism of two LRP ligands, activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein (Williams, S.E., Inoue, I., Tran, H., Fry, G. L., Pladet, M.W., Iverius, P.-H., Lalouel, J.-M., Chappell, D.A., and Strickland, D.K. (1994) J. Biol. Chem. 269, 8653-8658). The current study investigated the potential for this region of LPL to promote cellular catabolism of VLDL via LRP. A fragment comprising the C-terminal domain of LPL (designated LPLC) was expressed in bacteria and found to promote cellular binding, uptake, and degradation of normal human VLDL in a dose-dependent manner. These effects were present whether LPLC was added simultaneously with 125I-VLDL or was prebound to cell surfaces prior to the assay. Mutations involving Lys407, Trp393, Trp394, or deletion of the C-terminal 14 residues reduced the effects of LPLC. Three LRP-binding proteins, the receptor-associated protein, lactoferrin, and a polyclonal antibody against LRP, competed for 125I-VLDL degradation induced by LPLC. Heparin or heparinase treatment of cells prevented LPLC-induced 125I-VLDL catabolism. Thus, cell-surface proteoglycans play an important role in this pathway. Interestingly, either LPLC or LPL when added in excess could block LPL-induced 125I-VLDL degradation presumably by interacting directly with LRP. However, unlabeled VLDL could not prevent catabolism of 125I-labeled LPLC or LPL. These data show that cellular fates for VLDL versus LPLC or LPL are divergent. This is probably due to independent catabolism of the latter via cell-surface proteoglycans. In summary, these in vitro studies indicate that a fragment of LPL corresponding to the C-terminal domain mimics the native enzyme with respect to induction of VLDL catabolism via LRP. Because LPLC lacks the catalytic site of native LPL, these studies establish that lipase activity is not required for LRP-mediated lipoprotein catabolism.

The carboxyl-terminal domain of lipoprotein lipase binds to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and mediates binding of normal very low density lipoproteins to LRP.

Lipoprotein lipase (LPL) binds with high affinity to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and promotes binding, uptake, and degradation of normal triglyceride-rich lipoproteins in a process mediated by LRP (Chappell, D. A., Fry, G. L., Naknitx, M.A., Muhonen, L. E., Pladet, M. W., Iverius, P-H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). To localize the portion of LPL that is responsible for interacting with LRP, fragments of LPL were expressed in bacteria. A fragment of human LPL containing the COOH-terminal domain (residues 313-448, designated LPLC) which lacks the catalytic site was able to bind to LRP. Purified LRP bound specifically to microtiter wells coated with LPL or LPLC with KD values of 2.8 and 5 nM, respectively. The effects of several mutations of LPLC were tested. Mutation of Lys407 to Ala reduced the affinity of LPLC for LRP by approximately 10-fold. Like native LPL, LPLC prevented the binding of activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein to LRP and inhibited the internalization and degradation of activated alpha 2-macroglobulin and receptor-associated protein in cultured fibroblasts. LPLC also bound to 125I-labeled human normal triglyceride-rich lipoproteins and promoted their binding to purified LRP and to cultured cells. Mutation of Trp393 and Trp394 to Ala completely abolished the ability of LPLC to bind to lipoproteins, but had little effect on its interaction with LRP. These data indicate that the COOH-terminal domain of LPL may function both in binding lipoproteins and mediating their interaction with LRP.

Low density lipoprotein receptors bind and mediate cellular catabolism of normal very low density lipoproteins in vitro.

Very low density lipoproteins (VLDL) are heterogeneous, triglyceride-rich particles that are precursors of low density lipoproteins (LDL). Before conversion to LDL, the majority of VLDL are irreversibly cleared from plasma by uncertain mechanisms. To investigate one potential mechanism for VLDL clearance, we studied the ability of LDL receptors to mediate VLDL uptake in vitro. Small, intermediate, and large VLDL from normolipidemic humans were found to bind and undergo catabolism via LDL receptors on normal human fibroblasts. Binding to cell surfaces was up-regulated by lovastatin, an inducer of LDL receptors. Both LDL and a monoclonal antibody against the LDL receptor (IgG-C7) prevented binding of 125I-VLDL. Also, VLDL binding to mutant fibroblasts lacking LDL receptors was low. Thus, LDL receptors mediated VLDL interactions with cells. Binding affinity decreased near saturation, and the apparent number of high affinity sites decreased with increasing VLDL particle size. Because LDL receptors are small (M(r) 115,000) relative to VLDL (M(r) 9-24 x 10(6)) and are clustered in clathrin-coated pits, these findings suggest that steric hindrance becomes an important binding determinant near saturation and are consistent with a lattice model for LDL receptor-ligand interactions. The capacity for cellular catabolism of VLDL decreased with increasing particle size, consistent with a lattice model. The lattice model was also supported by differences between 125I-VLDL binding to cell surfaces and binding to partially purified LDL receptors in solid-phase assays in which steric constraints resulting from clustering in clathrin-coated pits are not present. In both cell-surface and solid-phase assays, VLDL bound via apoE, not apoB-100. Our studies establish that normal VLDL interact with LDL receptors and that steric hindrance due to crowding of particles on clustered LDL receptors is an important determinant of their binding and catabolism. These findings suggest that LDL receptors may participate in normal VLDL clearance in vivo.

Lipoprotein lipase induces catabolism of normal triglyceride-rich lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in vitro. A process facilitated by cell-surface proteoglycans.

Bovine milk lipoprotein lipase (LPL) induced binding, uptake, and degradation of 125I-labeled normal human triglyceride-rich lipoproteins by cultured mutant fibroblasts lacking LDL receptors. The induction was dose-dependent and occurred whether LPL and 125I-lipoproteins were added to incubation media simultaneously or LPL was allowed to bind to cell surfaces, and unbound LPL was removed by washing prior to the assay. Lipolytic modification of lipoproteins did not appear to be necessary for increased catabolism because the effect of LPL was not prevented by inhibitors of LPL's enzymatic activity, p-nitrophenyl N-dodecylcarbamate or phenylmethylsulfonyl fluoride. However, the effect was abolished by boiling LPL prior to the assay suggesting that major structural features of LPL were required. Also, LPL-induced binding to cells was blocked by an anti-LPL monoclonal antibody but not by antibodies that are known to block apolipoprotein E- or B-100-mediated binding to low density lipoprotein (LDL) receptors. This indicates that LPL itself mediated 125I-lipoprotein binding to cells. Cellular degradation of 125I-lipoproteins was partially or completely blocked by two previously described ligands for the LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP): activated alpha 2-macroglobulin (alpha 2M*), and the 39-kDa receptor-associated protein. These data implicated LRP as mediating LPL-induced lipoprotein degradation and were confirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. Furthermore, LPL promoted binding of 125I-lipoproteins to highly purified LRP in a solid-phase assay. Heparin or heparinase treatment of cells markedly decreased LPL-induced binding, uptake, and degradation of lipoproteins, but had no effect on catabolism of alpha 2M*. Thus, cell-surface proteoglycans were obligatory participants in the effects of LPL but were not required for LRP-mediated catabolism of alpha 2M*. Taken together, these in vitro findings establish that through interaction with cell-surface proteoglycans, LPL induces catabolism of normal human triglyceride-rich lipoproteins via LRP.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor binds and mediates catabolism of bovine milk lipoprotein lipase.

Lipoprotein lipase (LPL), the major lipolytic enzyme involved in the conversion of triglyceride-rich lipoproteins to remnants, was found to compete with binding of activated alpha 2-macroglobulin (alpha 2M*) to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. Bovine milk LPL displaced both 125I-labeled alpha 2M* and 39-kDa alpha 2M receptor-associated protein (RAP) from the surface of cultured mutant fibroblasts lacking LDL receptors with apparent KI values at 4 degrees C of 6.8 and 30 nM, respectively. Furthermore, LPL inhibited the cellular degradation of 125I-alpha 2M* at 37 degrees C. Because both alpha 2M* and RAP interact with LRP, these data suggest that LPL binds specifically to this receptor. This was further supported by observing that an immunoaffinity-isolated polyclonal antibody against LRP blocked cellular degradation of 125I-LPL in a dose-dependent manner. In addition, 125I-LPL bound to highly purified LRP in a solid-phase assay with a KD of 18 nM, and this binding could be partially displaced with alpha 2M* (KI = 7 nM) and RAP (KI = 3 nM). Taken together, these data establish that LPL binds with high affinity to LRP and undergoes LRP-mediated cellular uptake. The implication of these findings for lipoprotein catabolism in vivo may be important if LRP binding is preserved when LPL is attached to lipoproteins. If so, LPL might facilitate LRP-mediated clearance of lipoproteins.

Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.

Ligand size as a determinant for catabolism by the low density lipoprotein (LDL) receptor pathway. A lattice model for LDL binding.

Low density lipoproteins (LDL) are large (Mr = 2.5 x 10(6)) in comparison to LDL receptors (Mr = 115,000). Since most LDL receptors are clustered in coated pits, we tested the hypothesis that crowding of receptor-bound LDL particles would cause steric effects. The apparent affinity of LDL for receptors on cultured fibroblasts decreased near saturation causing concave-upward Scatchard plots. Both the higher and lower affinity components of binding were up-regulated by the cholesterol synthesis inhibitor, lovastatin, indicating that the entire binding curve was sterol-responsive. In contrast, neither component of LDL binding was present on lovastatin-treated or untreated null fibroblasts which are incapable of expressing LDL receptors. Therefore, the concave-upward Scatchard plots were entirely due to binding to LDL receptors. These results are consistent with a lattice model in which receptor-bound LDL are large enough to decrease binding to adjacent receptors. A lattice model implies that large LDL should produce steric effects at a lower receptor occupancy than should small LDL. This was tested using seven LDL fractions that differed in diameter from 20 to 27 nm. Fewer large than small LDL were bound to the cell surface at 4 degrees C and 37 degrees C, and fewer were internalized and degraded at 37 degrees C. Since large LDL bound via both apolipoprotein (apo) E and apoB100, receptor cross-linking could have caused fewer large LDL to be bound at saturation. However, when the potential for cross-linking was prevented by an apo-E-specific monoclonal antibody (1D7), the difference in binding by large versus small LDL was not eliminated; instead, it was exaggerated. Taken together, these results support a lattice model for LDL binding and indicate that steric hindrance associated with crowding of LDL particles on receptor lattices is a major determinant for catabolism by the LDL receptor pathway in vitro.

Ocular neovascularization. Tissue culture studies.

The proliferative activity of a number of intraocular fluids, bovine retinal extract, and normal serum (from humans and cynomolgus monkeys) was investigated by in vitro tissue culture studies, with the use of tritiated thymidine incorporation by the cultured endothelial cells of human umbilical veins. There was increased tritiated thymidine incorporation by (1) the aqueous, vitreous, and intraocular fluid (IOF) (which filled the eye after lensectomy and vitrectomy) removed from cynomolgus monkey eyes with iris neovascularization or with neovascular glaucoma (NVG) that developed after experimental retinal vein occlusion, (2) by aqueous and vitreous removed from human eyes with NVG or proliferative diabetic retinopathy; (3) by the serum, and (4) by the bovine retinal extract. However, tritiated thymidine incorporation was not increased by the normal aqueous, vitreous, or IOF.

Induction and amplification of T-lymphocyte proliferative responses to periodate and soybean agglutinin by human adult vascular endothelial cells.

Human mononuclear phagocyte (M phi) populations were compared to adult human endothelial cells (HEC) for their respective abilities to influence the proliferative responses of purified human T lymphocytes to the mitogenic agents Na-m-periodate (IO-4), soybean agglutinin (SBA), or allogeneic cells. HEC and M phi were both capable of inducing proliferative responses of allogeneic T lymphocytes in mixed-lymphocyte culture. Under low cell density culture conditions, purified T-lymphocyte proliferative responses to IO-4 or SBA could be restored by addition of syngeneic M phi or HEC. At higher cell density culture conditions, proliferation of T cells to IO-4 could be amplified more by HEC than M phi. T-lymphocyte proliferative responses to SBA were amplified by addition of HEC but were suppressed by addition of M phi. These findings indicate that human adult HEC are unique and potent accessory cells for T lymphocytes. Furthermore, these findings demonstrate that accessory cell functions of HEC can be discriminated from those of M phi.

Contribution of a platelet component to endothelial prostacyclin production despite inhibition of platelet cyclooxygenase activity.

Platelets aggregated with low concentrations of thrombin enhanced prostacyclin release from umbilical vein endothelium, which had an active cyclooxygenase. This result cannot be explained solely by an effect of thrombin on the endothelium or by transfer of the endoperoxide PGH2 from platelets to the endothelium. The quantity of prostacyclin released is sufficient to alter platelet adherence to umbilical vein endothelium in the presence of thrombin.

Role of the vascular endothelium.

A possible explanation for the non-thrombogenic effect of the endothelium is the presence of prostacyclin, (PGI2), the potent inhibitor of platelet aggregation and adherence, which is produced and released by the endothelium in response to various stimuli. Removal of PGI2 from the endothelium did not increase baseline platelet adherence, but did increase thrombin-induced platelet adherence from 4 to 60%. Additions of exogenous PGI2 at low concentrations reversed the enhanced thrombin-induced platelet adherence under these conditions. Although it is unlikely that prostacyclin is the sole factor regulating platelet adherence to the endothelium, it appears to play a major role in the interaction of platelets with components of the blood vessel wall. Conditions which predispose to adherence of platelets to the vessel wall may involve entrapment of tumor cells and lead to metastasis formation. Whether prostacyclin and other factors involved in the non-thrombogenic character of the vascular endothelium provide a significant defense against attachment of tumor cells is not known, but the potential for such a primary or secondary role clearly exists. In our studies, prostacyclin did not appear to influence the adherence of Raji lymphoma cells to the endothelium. Additional studies are indicated to correlate adherence of tumor cells to the vascular wall with their potential for formation of metastatic lesions.

Utilization of arachidonic and linoleic acids by cultured human endothelial cells.

When cultured human umbilical vein endothelial cells are supplemented with linoleic acid, the arachidonic acid content of the cellular phospholipids is reduced approximately 35%. Most of the fatty acid compositional change occurs during the first 24 h. One factor responsible for this effect is the inability of the endothelial cells to convert appreciable amounts of linoleic to arachidonic acid, due to a fatty acid delta 6-desaturase deficiency. By contrast, these endothelial cultures contain delta 5- and delta 9-desaturase activity and are able to elongate long-chain polyunsaturated fatty acids. The other factor that contributes to the decrease in arachidonic acid is that high concentrations of linoleic acid reduce the incorporation of arachidonate into cellular phospholipids. Stearic acid, a long-chain saturate, does not produce any reduction, whereas eicosatrienoic acid is an even more effective inhibitor than linoleic acid. In spite of the fact that high concentrations of these polyunsaturates produced inhibition, the endothelial cells were found to efficiently incorporate exogenous arachidonic acid into cellular phospholipids and triglycerides. This may serve to compensate for the inability of these cells to synthesize arachidonic acid from linoleic acid. These findings suggest that the endothelium obtains arachidonic acid from an extracellular source, that this cannot be provided in the form of linoleic acid and, in fact, that high concentrations of linoleic acid actually may interfere with the ability of the endothelium to maintain an adequate supply of intracellular arachidonic acid.