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The effects of thrombin (0.2 U/ml) and native (n-LDL), malondialdehyde-modified (MDA-LDL) and auto-oxidized (ox-LDL) low-density lipoproteins (20 micrograms of protein/ml) on platelet activation were evaluated in seven hyperlipidemic patients and compared to seven controls (fasting serum cholesterol 8.49 +/- 0.5 and 4.61 +/- 0.4 mM, respectively). Basal and thrombin-induced increases in platelet intracellular free calcium ion concentration ([Ca2+]i; fura-2) were similar in hyperlipidemic patients and controls (45 +/- 5 vs 42 +/- 3 and 635 +/- 51 vs 599 +/- 69 mM, respectively). n-LDL, MDA-LDL and ox-LDL increased basal [Ca2+]i (16, 36 and 81 percent, p < 0.01 between LDL-types), increases were consistently smaller in patients. There was an inverse relationship between LDL-induced responses and fasting serum LDL cholesterol as well as LDL/HDL ratio. In conclusion, modified LDL activated platelets to a greater extent than n-LDL, suggesting different types of LDL-receptors. Their agonistic effect was inversely related to the fasting serum lipid profile, suggesting that blunting of platelet responses to LDL could represent a protective mechanism in hyperlipidemic patients.

Non-insulin dependent diabetes mellitus (NIDDM) and hypertension are common diseases which are independently associated with insulin resistance/hyperinsulinemia, dyslysidemia, abnormalities of platelet function, and accelerated atherogenesis. The interaction of these independent risk factors is poorly understood. Recently, low density lipoprotein (LDL) receptors have been described, in platelets, and LDL elevates [Ca2+]i in these cells. In this study we have evaluated platelet [Ca2+]i responsiveness to LDL and arginine vasopressin (AVP) in NIDDM patients with (n = 28) and without (n = 13) concomitant hypertension, as well as in normal nondiabetic controls (n = 13). Platelet [Ca2+]i concentration-response curves to LDL for both NIDDM and hypertensive NIDDM were shifted significantly to the left when compared to the normotensive, nondiabetic controls. By contrast, no differences were seen in [Ca2+]i responses to 10 mumol/L AVP among any of the groups. To determine the possible role of hyperinsulinemia in this accentuated [Ca2+]i response to LDL, we measured basal and LDL-stimulated [Ca2+]i in platelets of normal volunteers after insulin treatment (0-100 mU/mL for 30 and 90 min). Insulin did not alter baseline or LDL-stimulated (150 mg/mL) platelet [Ca2+]i. Thus, an enhanced platelet [Ca2+]i response to LDL is characteristic of diabetes, independently of blood pressure. As such, it may also help to explain the enhanced platelet aggregation, endothelial dysfunction, and accelerated atherosclerosis of NIDDM.

HMG-CoA reductase catalyzes the synthesis of mevalonate, a crucial intermediate in the biosynthesis of cholesterol and non-sterol isoprenoid compounds essential for cell growth. The HMG-CoA reductase activity of the A549 tumor cell line is higher than that of normal human fibroblasts. This deregulation in mevalonate needs was not due to an alteration in the activated state of the enzyme by short-term regulation. We show that the HMG-CoA reductase in A549 cell line was subject to a multivalent feedback control. A high fraction (40%) of the reductase activity was devoted to non-sterol products. In contrast, normal fibroblasts had only 15-20% of the reductase activity that generated non-sterol products. We also show that cholesterol and at least one of the non-sterol products are necessary for optimal cell growth of A549 cells. Our data strongly suggest that A549 cells produce more non-sterol substances which may be related to increased requirements of mevalonate for upregulated cell growth.

Increased platelet activity on structurally and functionally impaired endothelium leads to a release of serotonin (5HT), thromboxane A2 and growth factors from platelets. These vasoactive substances may participate in the development of atherosclerosis and thrombovascular events. Platelet aggregatory response to 5HT increases with the height of blood pressure, age, smoking and plasma cholesterol levels. Lipoprotein fractions exert disparate effects an platelets. LDL-cholesterol amplifies 5HT-induced platelet activation whereas HDL-cholesterol inhibits the amplifying effect of LDL-cholesterol. These results indicate that different effects of HDL and LDL-cholesterol on atherosclerotic progression that where found in epidemiological and population studies take place at a cellular level and may represent one of the underlying cellular mechanisms of atherogenesis. This mechanism may be relevant to both atherogenesis and acute thrombovascular events and therefore may represent a target of pharmacological intervention. Effect of calcium antagonists on serotonin- and LDL-induced platelet aggregation were investigated. Isradipin and verapamil (but not diltiazem, amlodipin and felodipin) inhibited at therapeutic concentrations platelet aggregation induced by 5HT in vitro. Isradipin (but not diltiazem and felodipin) also inhibited the amplifing effect of LDL-cholesterol on 5HT-induced platelet aggregation in vitro. Effects of isradipin on 5HT-induced platelet activity as well as on the amplifying effect of LDL-cholesterol were observed also ex vivo. Calcium antagonists differ in their platelet inhibition potency, including their effects on platelet response to 5HT and LDL-cholesterol. These platelet effects of Calcium antagonists appear to be neither group- nor class-specific, but rather drug specific. Effect of isradipin may be relevant to platelet-mediated coronary vasospasm and to prevention of both atherogenesis and thrombogenesis.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.

A previous study in rats showed that even though probucol substantially lowers high density lipoprotein (HDL) levels, near-normal mass transport of HDL cholesterol esters (CE) to the liver is maintained by the induction of "selective" (direct) uptake of HDL CE. The present study describes a parallel result in cultured Hep G2 human hepatoma cells. Cells were preincubated in the presence or absence of probucol before measuring the uptake of doubly labeled HDL3 in the absence of probucol. Preincubation with probucol decreased the uptake of HDL3 particles (iodine-125-labeled N-methyltyramine cellobiose-apolipoprotein [125I-NMTC-apo] A-I uptake) but increased the uptake of [3H]cholesteryl oleyl ether in excess of 125I-NMTC-apo A-I (i.e., selective uptake) in a dose-dependent fashion. The reversibly cell-associated pool of CE tracer, a precursor for selective uptake, enlarged on probucol treatment, but the increase was not in proportion to the increase in selective uptake. HDL3 particle uptake decreased on probucol treatment. The decrease was evident after less than 20 minutes of probucol exposure and was maximal after 6 hours; in contrast, HDL3 CE selective uptake increased only after greater than 13 hours and had not reached a plateau after 20 hours. Thus, effects on particle uptake and selective uptake were dissociated in time.

To elucidate an anti-atherogenic nature of high density lipoprotein (HDL) at cellular level, its in vitro effect on macrophage foam cells was examined. Rat peritoneal macrophages were converted to foam cells by incubation with [3H]cholesterol-labeled acetylated LDL. HDL addition to these foam cells resulted in a reduction in cellular radioactive cholesteryl esters (CE) as well as its CE mass. The radioactive free cholesterol (FC) was similarly reduced with time, whereas its FC mass level was unaltered. Other lipoproteins such as very low density lipoprotein and low density lipoprotein also reduced the radioactive FC. However, their CE-reducing capacity was negligibly weak. These results suggest that (i) CE reduction is selective to HDL, (ii) FC transfer from plasma membrane to lipoprotein (cholesterol efflux) expressed by reduction in radioactive FC is not selective to HDL but occurs to other lipoproteins, (iii) the CE-reducing capacity of HDL became weaker when cellular binding of HDL was reduced by chemical modification with tetranitromethane or a chemical cross-linker, dithiobis-succinimidylpropionate, suggesting an importance of the specific binding in the HDL-mediated CE reduction. These in vitro results gave an experimental support to a definite role of HDL as an anti-atherogenic lipoprotein in vivo.

We have achieved in vivo expression of recombinant low-density-lipoprotein (LDL) receptors in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for the human disease familial hypercholesterolemia. A retroviral vector was constructed containing the human LDL receptor cDNA and was used to stably transduce primary skin fibroblasts from WHHL rabbits. The integrity and function of the introduced LDL receptor was established by immunoprecipitation, by a fluorescent LDL binding assay, and by the ability of the transduced cells to suppress 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase activity in response to exogenous cholesterol. Autologous transduced fibroblasts were reimplanted into donor rabbits; in vivo LDL receptor expression and the survival of the transduced cells were analyzed by immunohistochemistry and by LDL binding assays performed on cells recovered from the implants. LDL receptor-bearing cells could be identified on tissue sections and recovered from implants for up to four weeks. Total and LDL cholesterol levels decreased significantly after implantation of the transduced cells; however, control experiments indicated that the decreases were not mediated through the recombinant LDL receptor. While in vivo stable expression of recombinant LDL receptors in Watanabe rabbits is possible, consequent changes in lipid levels must be interpreted with caution. This system of site-specific in vivo expression of recombinant LDL receptors permits further evaluation of the role of LDL receptor-gene replacement in the therapy of hypercholesterolemia.

The aim of this research was to perform an in vivo study on the relationships between lipid oxide (LP), platelet aggregation and PAF-acetylhydrolase in a model using perfusion of cholesterol-rich LDL media diluted to physiological LDL-cholesterol concentration. Normal rabbits were infused with LDL (d 1.025-1.063 g/ml) extracted from rabbits previously fed either with standard food (I-LDL group), 1% cholesterol food (II-LDL group) or 1% cholesterol plus probucol (IV-LDL group). CU2+ modified II-LDL was also infused (III-LDL group). After dilution as above, LP increased significantly in III- and II-LDL media. After perfusion, LP significantly increased in III- and II-LDL groups as compared to baseline values and to control. Compared to the I-LDL group, PAF-acetylhydrolase and platelet aggregation significantly increased in III- and II-LDL groups. These data indicate the property of cholesterol- rich LDL to activate PAF-acetylhydrolase and enhance platelet aggregation, even when perfused through a medium containing a physiological LDL-cholesterol concentration.

Effects of various lipid components of low density lipoproteins (LDL) and serine on the regulation of UDP-Gal-beta 1-4-galactosyltransferase (GalT-2) activity have been investigated in normal proximal tubular (PT) cells. Addition of exogenous serine (0.1-0.75 mM), cholesterol (0-200 micrograms/ml medium), linoleic acid and oleic acid (0.1-0.75 mM) for 4 hr at 37 degrees C did not suppress the activity of GalT-2 in PT cells. Similarly, incubation of cells with glucosylceramide and lactosylceramide (25-50 micrograms/ml medium) did not alter GalT-2 activity in cells as compared to control. In contrast, palmitic acid (0-0.75 mM), phosphatidylethanolamine and sphingomyelin (0-200 micrograms/ml) stimulated GalT-2 activity by 20-36% as compared to control. Incubation of PT cells with D-alpha-dipalmitoyl phosphatidylcholine (0-200 micrograms/ml medium) also stimulated the activity of GalT-2, maximum stimulation (200%) occurring with 25 micrograms phosphatidylcholine/ml medium. However, at a higher concentration (200 micrograms/ml), the stimulation of the activity of GalT-2 was in the order of 27% compared to control. Dioleylphosphatidylcholine did not alter GalT-2 activity in PT cells. Thus, it is concluded that (i) various lipid components, sphingosine and serine present in LDL are not involved in the LDL-mediated suppression of GalT-2 activity in normal PT cells, and (ii) stringent structural requirements in the phosphatidylcholine molecule are necessary to exert a time and concentration dependent stimulation of GalT-2 activity.

The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane-associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL-levels increase, high extracellular LDL-levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.

1. The capacity of low-density lipoprotein-cholesterol (40 micrograms/ml) to stimulate both the phosphoinositide cycle and the dislocation of the phospholipid- and calcium-dependent protein kinase C activity from the cytosolic to the membranous compartment in human circulating lymphocytes has been studied. 2. Pretreatment of lymphocytes with pertussis toxin inhibited significantly the low-density-lipoprotein-induced formation of inositol trisphosphate. 3. The calcium-channel blockers verapamil, nifedipine and diltiazem, each in a concentration range from 1 mumol/l to 1 nmol/l, attenuated the low-density lipoprotein-induced formation of inositol trisphosphate in a dose-dependent manner. Similar results were observed when so-called non-specific calcium antagonists, such as flunarizine and molsidomine, were used. 4. The results suggest that low-density lipoprotein activates the phosphoinositide cycle in circulating lymphocytes through mechanisms sensitive to calcium blockers and pertussis toxin.

These studies were directed toward understanding the cellular actions of inhibitor drugs that affect steroidogenesis and cholesterol transport. We investigated the microfilament inhibitor cytochalasin-D, the microtubule inhibitor colchicine, the calmodulin antagonist trifluoperazine, and the inhibitor of acidic vesicle function nigericin. We found that all of these compounds caused dose-dependent inhibition of progesterone synthesis in the MA-10 cells. Each compound also inhibited (Bu)2cAMP-stimulated pregnenolone synthesis, indicating that each inhibited a fundamental process required for steroidogenesis. Each compound was next evaluated for inhibitory actions on cholesterol transport to and from the plasma membrane. On the basis of inhibitor sensitivity, two different categories of cholesterol transport were defined. Transport of newly synthesized or low density lipoprotein-derived cholesterol from the cell interior to the plasma membrane was inhibitor insensitive. Plasma membrane cholesterol internalization, however, was sensitive to all of the inhibitors and did not result because of any drug effect on the acyl-coenzyme-A-cholesterol acyl transferase. Cycling of cholesteryl ester-derived cholesterol through the plasma membrane appeared to occur before its use for steroidogenesis. Thus, inhibition of plasma membrane internalization would prevent utilization of both plasma membrane cholesterol and cholesteryl ester-derived cholesterol, the two major substrate sources for steroid hormone synthesis. Consistent with this interpretation was the finding that inhibition of plasma membrane cholesterol internalization by each inhibitor paralleled the inhibitor's effect on steroidogenesis.

Hyperlipidemia and hypertension are risk factors for cardiovascular diseases. To obtain insight into intracellular mechanisms underlying these phenomenon, the influence of low density lipoprotein (LDL) on the intracellular free calcium concentration, the intracellular pH, the calcium influx as well as proliferation rate of cultured vascular smooth muscle cells was studied. 1-15 micrograms/ml of LDL increased dose-dependently the concentration of intracellular free calcium and led to biphasic pH-shifts. 3-7 micrograms/ml of LDL augmented thymidine incorporation into cell DNA by 70% and enhanced calcium influx by 80%. LDL (1-15 micrograms/ml) clearly increased contractile response of aortic rings. The findings indicate that low concentrations of LDL may contribute to the pathogenesis of cardiovascular diseases by enhancing cell proliferation and vasoconstriction via changing intracellular calcium and intracellular pH.

Lipoproteins suppress several immune functions (mitogen-induced lymphoblast transformation, macrophage functions). Low density lipoprotein (LDL) is responsible for most of these effects. Diet may cause an elevation in LDL level. Therefore, we investigated the effect of cholesterol-rich diet upon the mitogen response of rats. The cholesterol and LDL-cholesterol level of rats was elevated after 8 days of diet. The blastogenic response of spleen cells separated by Ficoll-Paque gradient decreased. The response to phytomitogens 1, and 10 micrograms/ml phytohaemagglutinin (PHA) and 10 micrograms/ml concanavalin A (Con A) was measured by thymidine incorporation after 3 days of culture. The decrease of blastogenic response was more pronounced at the higher doses (10 micrograms/ml PHA, and 10 micrograms/ml Con A). For the lymphocyte cultures that did not contain autologous (rat) serum, we suggest that suppressed response was caused by an alteration of either in the membrane or in the metabolism of cells participating in the mitogen response. The decreased amount of LDL receptors and a derangement in intracellular cholesterol biosynthesis are supposed. The clinical implications of the immunosuppressive effect of dietary cholesterol is discussed.

We studied the biochemical and biological properties of plasma lipoproteins taken from blood derived from either the aorta or femoral vein of patients with normal coronary arteriography. There were no significant differences in concentrations of cholesterol, triglycerides, apoprotein A-1 and apoprotein B derived from either source. The cholesterol content of very low density lipoprotein (VLDL) and high density lipoprotein (HDL) was similar in both aortic and venous blood. The low density lipoprotein (LDL) concentration, however, was significantly higher in the aortic blood sample. Arterial LDL significantly enhanced in vitro platelet aggregation when compared to venous LDL. (p less than 0.02). When incubated with mouse peritoneal macrophages (MPM) arterial LDL and VLDL caused an increased cholesterol accumulation and enhanced cholesterol esterification within these macrophages. The venous lipoproteins had little effect. The differences noted in the arterial lipoproteins in composition and biological function when compared to venous lipoproteins might be related to the much higher incidence of atherosclerosis found in the arterial tree.

The influence of low density lipoproteins (LDL)-cholesterol (1.69 +/- 0.08 mg/ml) and of high density lipoproteins (HDL)-cholesterol (0.62 +/- 0.09 mg/ml), respectively, on the levels of 6-oxo-PGF1 alpha and TXB2 in the perfusates of rat aortas perfused under pulsatile pressure was studied. After passage through the aortas the concentration of LDL-cholesterol was significantly decreased in the perfusates, whereas the HDL-cholesterol level was unchanged. In comparison to controls, perfused with lipoprotein free solution, the concentration of 6-oxo-PGF1 alpha was significantly decreased more than 50% by LDL and the TXB2 level was enhanced significantly by approximately 50%. This results in a significant rise of the TXB2/6-oxo-PGF1 alpha ratio in the perfusates. HDL did not significantly change the ratio of these eicosanoids in the perfusates. These results suggest that this concentration of LDL enhanced the formation of the proaggregatory TXA2 and decreased the formation of the antiaggregatory PGI2 in rat aortas perfused under pulsatile pressure. The proaggregatory action of an elevated level of LDL during the development of atherosclerosis in men may be also mediated by changes in the metabolism of eicosanoids of the vessel wall.

We have shown that NS-1 mouse myeloma cells, but not NS-1 hybridomas, required human low density lipoprotein (LDL) for survival and growth in serum-free cell culture (Kawamoto et al., 1983). Here we have further defined the lipid requirement of NS-1 cells by demonstrating that LDL could be replaced by cholesterol complexed with carrier bovine serum albumin (BSA). Cholesterol was the active component of this complex since BSA alone did not promote NS-1 survival or growth. Cholesterol was an absolute requirement of these cells, and it could not be replaced by mevalonolactone. In contrast to NS-1 cells a related mouse myeloma cell line, Sp2/0, did not require cholesterol or other lipids for growth in vitro. Finally, we propose that cholesterol-deficient medium may be an effective alternative to HAT medium for selecting NS-1 hybridomas.

A biochemical correlation between vascular cholesterol metabolism and eicosanoid biosynthesis has not been fully elucidated. To assess the effects of cholesteryl ester (CE) accretion on eicosanoid synthesis, we studied eicosanoid metabolism in cultured rabbit aortic smooth muscle cells (SMC) following lipid-enrichment by incubation with cationized LDL (cLDL). SMC exposed to cLDL synthesized 50% less immunoreactive 6-keto-PGF1 alpha than untreated cells when exposed to the calcium ionophore, A-23187. In addition, cLDL-treatment reduced arachidonate acid (AA)-induced prostacyclin (PGI2) production sevenfold. Components of cLDL decreased eicosanoid biosynthesis in the following rank-order: linoleate greater than cholesterol greater than apo-cLDL. Lipid-enriched cells incorporated amounts of [1-14C]AA into phosphatidylcholine and phosphatidylethanolamine equal to control cells, but subsequent exposure to ionophore released significantly less radioactivity as free arachidonate (AA), with proportionally less conversion to eicosanoids. Ionophore released equivalent amounts of AA from all phospholipids, suggesting specificity for uptake, but not release of AA by cellular phospholipases. Cells enriched in CE had an eightfold decrease in percentage of phospholipid-derived AA relative to linoleate as compared to controls. Taken together, our data demonstrate that SMC metabolism of cLDL leads to cholesterol and CE accretion concomitant with diminished production of eicosanoids. Potential mechanisms for this effect include competitive inhibition of eicosanoid production by linoleate derived from LDL, direct inhibition of phospholipase A2 activity by cholesterol, and decrease in cyclooxygenase activity. These findings may have pathophysiological significance in that a reduction in PGI2 synthetic capacity of arterial SMC may exacerbate CE deposition since PGI2 promotes intracellular CE hydrolysis.

The role of bovine HDL and LDL in supporting corticotropin-stimulated steroidogenesis has been investigated, using acutely dispersed zona fasciculata cells. Using a dose of corticotropin sufficient to maximally stimulate steroidogenesis (in the absence of lipoproteins) both HDL and LDL increased steroidogenesis in a dose-dependent manner. At higher concentrations of lipoprotein, HDL caused approx 3-fold greater increase in steroid production than did LDL. Taken with the knowledge that HDL is the major cholesterol carrier in bovine serum, these findings suggest that in cattle HDL is a major source of cholesterol for steroidogenesis.

In familial hypercholesterolemia, structural and functional abnormalities of the receptor for low-density lipoprotein (LDL) lead to hypercholesterolemia and premature atherosclerosis. We have developed a simplified method to identify LDL-receptor defects in peripheral-blood lymphocytes. When lymphocytes are cultured in lipoprotein-depleted medium and endogenous sterol biosynthesis is suppressed with mevinolin, mitogen-stimulated proliferation of lymphocytes is dependent on an exogenous source of cholesterol. Whereas a small concentration of supplemental LDL cholesterol (3 to 4 micrograms per milliliter) permits a maximal response in normal lymphocytes, even high concentrations (10 to 50 micrograms per milliliter) are unable to support the proliferation of lymphocytes from patients with homozygous familial hypercholesterolemia. Thus, functional LDL receptors are necessary to allow lymphocyte proliferation in these cultures. The response of lymphocytes from patients with hyperlipidemia not caused by defective LDL receptors was like that of normal cells. In contrast, the response of lymphocytes from patients with heterozygous familial hypercholesterolemia was intermediate between that of homozygotes and that of normal or hyperlipidemic controls. Our method can therefore be used to identify persons who are heterozygous for abnormalities of LDL receptors.

We studied the effects of lipoprotein-derived cholesterol on the ACTH-induced differentiation of cultured fetal rat adrenocortical cells. For this purpose human plasma high-density lipoprotein3 (HDL3) or low-density lipoprotein (LDL) was added to culture media devoid of cholesterol, and thereafter the morphological changes in cells were monitored and the amounts of steroids synthesized were measured. It could be demonstrated that, ultrastructurally, upon ACTH-stimulation the adrenocortical cells differentiated into fasciculata-like cells even in the absence of lipoproteins in the culture medium. The addition of either HDL3 or LDL caused an increase in the number and size of cytoplasmic lipid droplets suggesting uptake and deposition of lipoprotein-derived cholesterol into the differentiating cells. The amount of steroids secreted from cells differentiating in media devoid of cholesterol was only half that observed in cells differentiating in serum-supplemented medium. Addition of either HDL3 or LDL increased the ACTH-stimulated steroid synthesis to the levels observed in serum-supplemented medium. This study demonstrates that both HDL3 and LDL are able to provide cholesterol for steroid synthesis accompanying the ACTH-induced differentiation of fetal rat adrenocortical cells.

In FH, abnormalities of the gene encoding the receptor for LDL lead to hypercholesterolemia and premature atherosclerosis. A method to identify LDL receptor defects using peripheral blood lymphocytes has been developed. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. When exogenous sterol was provided as a plasma lipoprotein, LDL receptor-mediated interaction with apolipoprotein-B or -E was essential for the provision of cholesterol to normal human lymphocytes. Thus, functional LDL receptors were necessary to permit proliferation of normal lymphocytes in these cultures. Lymphocytes from patients heterozygous for abnormalities in the LDL receptor gene can be distinguished from normal lymphocytes by their diminished functional LDL receptor activity. Of interest, following treatment with plasma cholesterol-lowering agents, functional lymphocyte LDL receptor activity normalized in some but not all patients with heterozygous FH, whereas plasma LDL cholesterol levels decreased in all patients. These results suggest that therapy with plasma cholesterol-lowering agents can lead to increased expression of LDL receptors by lymphocytes in the majority of patients with heterozygous FH. The failure of some heterozygous FH patients to increase functional LDL receptor activity after prolonged therapy indicates that there is heterogeneity in these patients despite a similar capacity of the therapy to decrease plasma LDL cholesterol. Variability in the expression of the normal LDL receptor gene in individual T lymphocytes may account for some of these findings.

A hypothesis of Gryglewski et al. explains the correlation between increased level of LDL and development of atherosclerosis by inhibition of PGI2 synthesis by increased peroxide content of LDL. The aim of the present paper was to examine this hypothesis. The major results are: 1) Preparation of LDL in the presence of .02% butylated hydroxytoluene did not reduce the lipid peroxide content of LDL from men and women and not change the inhibition or stimulation of the in vitro biosynthesis of PGI2 by LDL isolated from blood of men or women, respectively. 2) In the LDL and HDL, respectively, of healthy men we found nearly the same lipid peroxide levels (nmole malondialdehyde (MDA)/mg lipoprotein-cholesterol) as in the lipoproteins of male patients with hyperlipidemia type IIa or IV, but the peroxide concentration is three times higher in HDL as in LDL. 3) LDL isolated from healthy men inhibited in dose dependent fashion the generation of PGI2 from PGH2 by aortic microsomes whereas LDL from premenopausal women stimulated PGI2 formation even calculated as LDL lipid peroxides (in nM MDA/ml). The results call into question the hypothesis that diminished PGI2 formation by atherosclerotic vessels is related to inhibition of PGI2 synthetase by lipid peroxides present in LDL in the lesions. A new working hypothesis is presented that also the fatty acid pattern and the lipid class composition in the LDL are important for their influence on the PGI2 formation.