The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction.

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.

Oxidized LDL-induced injury and apoptosis in atherosclerosis. Potential roles for oxysterols.

The cell injury caused by oxidized lipoproteins was among the first findings that led to the theory that it is the oxidation of low-density lipoprotein (LDL), not just LDL concentration, that leads to arterial disease. Voluminous studies have now revealed that oxidized lipoproteins and their constituents can induce numerous effects on cells that can be construed to be atherogenic. Cell injury is but one of these, and it is these injurious effects that are the focus of this brief review. Cell injury and death appear to play multiple roles in lesion development and the toxic lipid constituents of oxidized lipoproteins, including a variety of oxysterols, are candidates for the in vivo effectors of this cytotoxicity. Recent studies have focused on the mechanisms of oxidized lipoprotein-induced cell death, whether the cells die by apoptosis or necrosis, and the identities of the toxins that induce injury. Understanding the roles of these agents in lesion development could lead to therapies that modulate cell death and inhibit lesion formation.

Smooth muscle cell surface tissue factor pathway activation by oxidized low-density lipoprotein requires cellular lipid peroxidation.

Tissue factor, which is expressed in vascular lesions, increases thrombin production, blood coagulation, and smooth muscle cell proliferation. We demonstrate that oxidized low-density lipoprotein (LDL) induces surface tissue factor pathway activity (ie, activity of the tissue factor:factor VIIa complex) on human and rat smooth muscle cells. Tissue factor messenger RNA (mRNA) was induced by oxidized LDL or native LDL; however, native LDL did not markedly increase tissue factor activity. We hypothesized that oxidized LDL mediated the activation of the tissue factor pathway via an oxidant-dependent mechanism, because antioxidants blocked the enhanced tissue factor pathway activity by oxidized LDL, but not the increased mRNA or protein induction. We separated total lipid extracts of oxidized LDL using high-performance liquid chromatography (HPLC). This yielded 2 major peaks that induced tissue factor activity. Of the known oxysterols contained in the first peak, 7alpha- or 7beta-hydroxy or 7-ketocholesterol had no effect on tissue factor pathway activity; however, 7beta-hydroperoxycholesterol increased tissue factor pathway activity without induction of tissue factor mRNA. Tertiary butyl hydroperoxide also increased tissue factor pathway activity, suggesting that lipid hydroperoxides, some of which exist in atherosclerotic lesions, activate the tissue factor pathway. We speculate that thrombin production could be elevated via a mechanism involving peroxidation of cellular lipids, contributing to arterial thrombosis after plaque rupture. Our data suggest a mechanism by which antioxidants may offer a clinical benefit in acute coronary syndrome and restenosis.

Relationship of molecular structure to the mechanism of lysophospholipid-induced smooth muscle cell proliferation.

We previously reported that oxidized low-density lipoprotein and one of its constituents, lysophosphatidylcholine (lysoPC), caused smooth muscle cell proliferation that was inhibitable by vitamin E and by a neutralizing antibody against basic fibroblast growth factor-2 (FGF-2). We now show that the mitogenic activity of lysolipids is highly dependent on structure. Phospholipids with palmitoyl fatty acid and phosphocholine induced DNA synthesis optimally. Shorter and longer fatty acids were significantly less potent, as were phosphoserine and phosphoethanolamine head groups. Structurally related phospholipids [platelet-activating factor (PAF) and lysoPAF] were also mitogens and acted via an analogous FGF-2-dependent, vitamin E-inhibitable mechanism. The mechanism of lysoPC stimulation was distinct from that of another phospholipid mitogen, lysophosphatidic acid (lysoPA), in that lysoPC stimulation was not pertussis toxin inhibitable. Furthermore, lysoPA stimulation was not inhibitable by vitamin E. Despite its distinct cellular pathway for stimulation, lysoPA also ultimately led to FGF-2 release. Our data show that specific structural attributes of lysoPC, PAF, and lysoPAF enable these agents to mediate smooth muscle cell release of FGF-2, which in turn stimulates proliferation.

Lysophosphatidylcholine-induced cellular injury in cultured fibroblasts involves oxidative events.

Lysophosphatidylcholine (lysoPC), formed during LDL oxidation and located within atherosclerotic plaques, induces numerous cellular responses, but via unknown mechanisms. Cellular events involved in sublethal lysoPC-induced injury were examined because these are relevant to mechanisms by which lysoPC alters cell behavior. LysoPC evoked transient membrane permeabilization in fibroblasts within 10 min. Cells underwent reversible rounding within 2 h, returning 3 h later to grossly normal appearance and a normal response to growth stimulation. We asked whether this sublethal permeabilization resulted from physical perturbation of the plasma membrane or if it required cellular events. LysoPC induced leakage of fluorescent dye from unilamellar phospholipid vesicles, suggesting physical membrane perturbation was a significant contributor. To characterize this further we increased the cholesterol content of cells and vesicles to stabilize membranes, and found decreased lysoPC-induced permeabilization in both cell and cell-free systems as cholesterol levels increased. Interestingly, vitamin E, a known antioxidant, blunted lysoPC-induced permeabilization and morphological changes in cells. Thus, lysoPC appeared to cause an unexpected oxidant stress-dependent enhancement of cell injury. To confirm this, several structurally distinct antioxidants, including N, N'-diphenyl-1,4-phenylenediamine, Desferal, Tiron, and 4-hydroxy TEMPO, were applied and these also were inhibitory. Oxidant stress was observed by a lysoPC-induced increase in fluorescence of 5- and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, an intracellular marker of reactive oxygen species. Lysophosphatidylethanolamine (lysoPE) caused qualitatively similar morphological changes to cells and induced permeabilization, but injury by lysoPE was not inhibited by antioxidants. These data suggest that generation of intracellular reactive oxygen species follows lysoPC-induced plasma membrane destabilization and that this lysoPC-specific oxidant stress enhances cell injury. This intracellular oxidant stress in response to lysoPC may be an integral part of the multiple influences lysoPC has on gene expression and cell function.

Regulation of cell growth by oxidized LDL.

The first reports of the influences of oxidized LDL (oxLDL) on cell function pertained to negative effects on cell growth-growth arrest, injury, and toxicity. Since these studies, it has become apparent that sublethal levels of oxLDL cause some, but not all, cells to proliferate. This review highlights the growth-promoting effects of oxLDL rather than its inhibitory or injurious effects. Smooth muscle cells (SMCs) and monocyte-macrophages proliferate after exposure to oxLDL; endothelial cells do not. Scavenger receptors are involved in the proliferative effects on monocyte-macrophages, whereas the effects of oxLDL on SMCs appear to be receptor independent. Lysophosphatidylcholine (lysoPC), and structurally related lipids are among the growth-promoting constituents of oxLDL. OxLDL exerts at least a part of its effects by inducing expression or causing the release of growth factors. OxLDL (or lysoPC) can cause the release of basic fibroblast growth factor (bFGF) from SMCs; oxLDL (or lysoPC) can induce heparin binding EGF-like growth factor (HB-EGF) synthesis and release from macrophages. An imposing array of changes in cytokine and growth factor expression and/or release can be imposed by oxLDL on a wide variety of cell types. These effects and the studies probing the cell signaling events leading to them are described.

The oxidative modification hypothesis of atherogenesis: an overview.

The literature relating lipid and lipoprotein oxidation to atherosclerosis has expanded enormously in recent years. Papers on the "oxidative modification hypothesis" of atherogenesis have ranged from the most basic studies of the chemistry and enzymology of LDL oxidation, through studies of the biological effects of oxidized LDL on cultured cells, and on to in vivo studies of the effects of antioxidants on atherosclerosis in animals and humans. The data in support of this theory are mounting but many key questions remain unanswered. For example, while it is generally agreed that LDL undergoes oxidation and that oxidized LDL is present in arterial lesions, it is still not known how and where LDL gets oxidized in vivo nor which of its many biological effects demonstrable in vitro are relevant to atherogenesis in vivo. This brief review is not intended to be comprehensive but rather to offer a perspective and a context for this Forum. We discuss the strengths and weaknesses of each line of evidence, try to identify areas in which further research is needed, assess the relevance of the hypothesis to the human disease, and point to some of the potential targets for therapy.

Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum.

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.

Native and oxidized low density lipoprotein induction of tissue factor gene expression in smooth muscle cells is mediated by both Egr-1 and Sp1.

Tissue factor, in association with factor VIIa, initiates the coagulation cascade. We studied the influences of two pathophysiological stimuli, native (unmodified) and oxidized low density lipoprotein, on tissue factor gene expression in a cell important in vascular remodeling and vascular diseases, the smooth muscle cell. Our results demonstrated that both lipoproteins significantly induced tissue factor gene expression in rat aortic smooth muscle cells; oxidized low density lipoprotein was slightly more potent. Both lipoproteins increased tissue factor mRNA in a concentration- and time-dependent manner. Results from nuclear run-on assays and mRNA stability experiments indicated that increased tissue factor mRNA accumulation in response to the lipoproteins was principally controlled at the transcriptional level. By using lipid extracts of low density lipoprotein or methylation of the intact lipoprotein to block receptor recognition, we showed that this lipoprotein induced tissue factor mRNA via both receptor-independent and receptor-augmented pathways. Transfection studies using a series of deleted tissue factor promoters revealed that a -143- to +106-base pair region of the rat tissue factor promoter contained regulatory elements required for lipoprotein-mediated induction. Electrophoretic mobility shift assays showed that the binding activities of the transcription factor Egr-1, but not Sp1, were markedly elevated in response to these lipoproteins. Transfection of site-directed mutants of the tissue factor (TF) promoter demonstrated that not only Egr-1 but also Sp1 cis-acting elements in the TF (-143) promoter construct were necessary for optimal TF gene induction. Our data show for the first time that both low density lipoprotein and oxidized low density lipoprotein induce tissue factor gene expression in smooth muscle cells and that this tissue factor gene expression is mediated by both Egr-1 and Sp1 transcription factors.

LDL increases inactive tissue factor on vascular smooth muscle cell surfaces: hydrogen peroxide activates latent cell surface tissue factor.

BACKGROUND: Tissue factor, which is required for the initiation of the extrinsic coagulation cascade, is known to be upregulated in cells within atherosclerotic lesions, including smooth muscle cells. Tissue factor expression on the smooth muscle cell surface could be of pathological significance as a contributor to plaque growth, thrombus formation, and the acute coronary syndrome after plaque rupture. METHODS AND RESULTS: In this study, we show that LDL increased tissue factor mRNA and cell surface protein in smooth muscle cells without a marked increase in surface tissue factor activity. Hydrogen peroxide activated tissue factor on the cell surface but did not increase tissue factor mRNA or cell surface protein. Sequentially added LDL and hydrogen peroxide increased mRNA, cell surface protein, and activity; surface activity was greater than that observed with hydrogen peroxide alone. The action of hydrogen peroxide did not involve a regulatory mechanism associated with the cytoplasmic tail of tissue factor because a truncated tissue factor lacking the cytoplasmic tail was activated by hydrogen peroxide. CONCLUSIONS: These results suggest a novel 2-step pathway for increased tissue factor activity on smooth muscle cell surfaces in which lipoproteins regulate synthesis of a latent tissue factor and oxidants activate the protein complex.

Arterial injury by cholesterol oxidation products causes endothelial dysfunction and arterial wall cholesterol accumulation.

Cholesterol oxidation products (ChOx) have been reported to cause acute vascular injury in vivo; however, the pharmacokinetics of ChOx after administration and the mechanisms by which they cause chronic vascular injury are not well understood. To further study the pharmacokinetics and atherogenic properties of ChOx, New Zealand White rabbits were injected intravenously (70 mg per injection, 20 injections per animal) with a ChOx mixture having a composition similar to that found in vivo during a 70-day period. Total ChOx concentrations in plasma peaked almost immediately after a single injection, declined rapidly, and returned to preinjection levels in 2 hours. After multiple injections, the ChOx concentrations rose gradually to levels 2- to 3-fold above baseline levels, increasing mostly in the cholesteryl ester fraction of LDL and VLDL. Rabbit serum and the isolated LDL/VLDL fraction containing elevated ChOx concentrations were cytotoxic to V79 fibroblasts and rabbit aortic endothelial cells. At the time of killing, cholesterol levels in the aortas from ChOx-injected rabbits were significantly elevated despite the fact that plasma cholesterol levels remained in the normal range. In addition, aortas from the ChOx-injected rabbits retained more 125I-labeled horseradish peroxidase, measured 20 minutes after intravenous injection. Transmural concentration profiles across the arterial wall also showed increased horseradish peroxidase accumulation in the inner half of the media from the thoracic aorta in ChOx-injected rabbits. In conclusion, ChOx injection resulted in accumulation of circulating ChOx and induced increased vascular permeability and accumulation of lipids and macromolecules. This study reveals that even under normocholesterolemic conditions, ChOx can cause endothelial dysfunction, increased macromolecular permeability, and increased cholesterol accumulation, parameters believed to be involved in the development of early atherosclerotic lesions.

Quantitative evaluation of local drug delivery using the InfusaSleeve catheter.

BACKGROUND: Restenosis is the most common long-term complication after angioplasty. Local delivery of pharmacologic agents at the site of angioplasty holds promise as a means of achieving higher concentrations of drug in the arterial wall than can be obtained by systemic infusion. In this study, a novel local drug delivery catheter system, the InfusaSleeve catheter, was evaluated in a porcine coronary balloon injury model. The purpose of the study was to evaluate the efficacy of solute transfer to the arterial wall and the influence of varying supporting angioplasty balloon pressure. METHODS AND RESULTS: Ten pigs (total of 22 arterial segments) underwent overstretch balloon injury (artery/balloon ratio 1:1.29) with a standard angioplasty balloon. In 7 animals (16 arterial segments) horseradish peroxidase (HRP; 10 mg/ml) was administered locally after injury, by tracking the local infusion catheter as a sheath over the angioplasty balloon to the intended site of arterial drug delivery. Supporting angioplasty balloons were inflated to one of the three different pressures. In 3 pigs HRP (10 mg/ml) was administered intravenously. No significant arterial injury caused by the local delivery device was evident on histological examination (disruption of the internal lamina elastica, arterial media, or thrombosis). Radial concentrations of the HRP reaction product in the first 150 microns of the arterial wall were quantified against known standards by measurement of light transmission through tissue sections. Mean HRP concentrations were not significantly different from those obtained by intravenous infusion using a supporting pressure of 1 atm or a supporting pressure of 3 atm of the underlying angioplasty balloon. However, a supporting pressure of 6 atm resulted in a 6-fold greater mean HRP concentration in the arterial wall than that which could be achieved by systemic administration of an equal volume of tracer (P < 0.001). CONCLUSION: Thus solute can be delivered throughout the coronary media by the InfusaSleeve, with the magnitude of wall uptake related to support pressure. Local delivery at 6 atm support pressure produced substantially greater uptake than did systemic delivery.

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.

Macromolecular transport in the arterial intima: comparison of chronic and acute injuries.

Hypertension is a known risk factor for the development of atherosclerosis, which is characterized by the abnormal accumulation of low-density lipoprotein and other plasma-borne macromolecules. The goal of this study was to measure accumulation of a plasma-borne macromolecular marker, horseradish peroxidase (HRP; 44 kDa), in the aortic intima and media of chronically hypertensive rats. HRP transport in 2-yr-old spontaneously hypertensive rats (SHR) was compared with that in age-matched Wistar-Kyoto rats (WKY) under conditions in which blood pressures were not significantly different during the 15-min HRP circulation. Intimal accumulation and medial HRP concentration profiles were obtained from methacrylate-embedded sections after reaction with 3,3'-diaminobenzidine and H2O2. Data were analyzed using a mathematical model of macromolecular transport to quantify the permeabilities of endothelium and internal elastic lamina (IEL). Chronic hypertension increased endothelial permeability without a change in IEL permeability. An apparent convective flux of HRP into the intima of SHR raised intimal HRP to a concentration higher than that of HRP in the plasma. Our results suggest that the intimal accumulation of plasma-borne macromolecules from pressure-driven convection is normally minimized by an intact endothelium. Similar changes resulted from acute injury by lipopolysaccharide, suggesting endothelial injury could account for transport changes associated with hypertension. After either chronic or acute endothelial damage, transport of macromolecules into the intima increases, but the IEL continues to retard transport of macromolecules beyond the intima, resulting in increased intimal accumulation.

Oxidized LDL mediates the release of fibroblast growth factor-1.

Fibroblast growth factor-1 (FGF-1) and lipoproteins play an important role in atherogenesis. In the present study, we explored a possible mechanism by which abnormal lipid metabolism could be linked to the proliferative aspects of the disease. We tested oxidized LDL (oxLDL) as a possible pathophysiological mediator of the release of FGF-1, using FGF-1-transfected mouse NIH 3T3 cells and FGF-1-transfected rabbit smooth muscle cells, and compared it with the release caused by elevated temperature. Immunoblot analysis showed that oxLDL induced the release of FGF-1 in a concentration-dependent manner from 10 to 100 micrograms/mL. The effect correlated with the extent of oxidative modification of LDL and was maximal within 4 hours of exposure of cells to oxLDL. In contrast to the temperature stress-induced FGF-1 secretion pathway, FGF-1 released in response to oxLDL (1) appeared in the conditioned medium as a monomer, (2) appeared independently of the presence of either actinomycin D or cycloheximide, and (3) was neither enhanced nor inhibited by brefeldin A. We did not detect cell loss, significant morphological changes, changes in growth characteristics, or other indications of lethal toxicity in oxLDL-treated cells. Although the level of lactate dehydrogenase activity was elevated after oxLDL exposure, the calculations showed that > 90% of the FGF-1 was released by viable cells. We propose that oxLDL-induced FGF-1 release is mediated by sublethal and apparently transient changes in cell membrane permeability. In the environment of an atherosclerotic lesion, oxLDL-induced FGF-1 release may be among the mediators of endothelial and smooth muscle cell proliferation.

Exogenous oxidized low-density lipoprotein injures and alters the barrier function of endothelium in rats in vivo.

Oxidation converts low-density lipoprotein (LDL) into a cytotoxin in vitro. Oxidized LDL exists in vivo in atherosclerotic lesions and possibly in plasma. Many cell functions are altered in vitro by oxidized LDL, but few have been examined in vivo. To test whether oxidized LDL could injure endothelial cells and alter endothelial permeability to macromolecules in vivo, we infused oxidized LDL, native LDL, or their solvent intravenously into rats. Subsequently, endothelial cell injury and proliferation were measured, and the transport into the aorta wall of the macromolecule horseradish peroxidase (HRP) was quantified. Transport data were analyzed using mathematical models of macromolecular transport; parameters were estimated by optimally fitting model-predicted HRP concentrations to experimental data. Compared with native LDL or solvent control infusion, oxidized LDL infusion increased (1) the number of injured aortic endothelial cells fivefold to sixfold at 36 hours, (2) proliferation of endothelial cells at 48 hours, (3) intimal and medial accumulations of HRP twofold to threefold at 48 hours, and (4) the permeability coefficient of the endothelium to HRP fourfold to fivefold at 48 hours. Hence, oxidized LDL administered in vivo can injure the endothelium, despite the presence of endogenous antioxidants, compromising the function of the endothelium as a permeability barrier.

Roles of multiple oxidized LDL lipids in cellular injury: dominance of 7 beta-hydroperoxycholesterol.

The relative toxicities of several lipid oxidation products formed on oxidized LDL, their presence on oxidized LDL, and potential mechanisms of cell injury compared to oxidized LDL were examined. Toxicities to fibroblasts, with lipoprotein-deficient serum supplementation, were: 7 beta-hydroperoxycholesterol > 7 beta-hydroxycholesterol = 4-hydroxynonenal > 7-ketocholesterol > 5 alpha, 6 alpha-epoxycholesterol. Lysophosphatidylcholine was only significantly cytotoxic in the absence of lipoprotein-deficient serum. Without serum, relative toxicities were: 7 beta-hydroperoxycholesterol > lysophosphatidylcholine > 4-hydroxynonenal > 7 beta-hydroxycholesterol. Similar relative potencies were observed in smooth muscle and endothelial cell cultures. 7 beta-Hydroperoxycholesterol accumulated on oxidized LDL to greater amounts than other oxysterols and 4-hydroxynonenal, but less than lysophosphatidylcholine. Cell injury by 7 beta-hydroperoxycholesterol and oxidized LDL was inhibitable by antioxidants but not by exogenous cholesterol or cycloheximide. In contrast, a) toxicities by 7 beta-hydroxycholesterol, 7-ketocholesterol, 5 alpha, 6 alpha-epoxycholesterol, and 4-hydroxynonenal were not inhibited by antioxidants; b) 7 beta-hydroxycholesterol and lysophosphatidylcholine toxicities were inhibited by exogenous cholesterol; and c) 7 beta-hydroxycholesterol toxicity was inhibited by cycloheximide. Injury by lysophosphatidylcholine was reduced by vitamin E and not affected by altering the cellular exposure to selenium; reduced selenium enhanced toxicity by oxidized LDL and 7 beta-hydroperoxycholesterol. The high relative toxicity of 7 beta-hydroperoxycholesterol, the level of its accumulation on oxidized LDL, and its mechanism of action similar to oxidized LDL suggest that it is the compound predominantly responsible for oxidized LDL induced cytotoxicity.

Angiotensin-(1-7) inhibits vascular smooth muscle cell growth.

Although angiotensin II (Ang II) and the heptapeptide Ang-(1-7) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(1-7) released prostaglandins. We now report that Ang II and Ang-(1-7) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(1-7) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(1-7) depended on the concentration of the heptapeptide over the range of 1 nmol/L to 1 mumol/L, with a maximal inhibition of 60% by 1 mumol/L Ang-(1-7). Ang-(1-7) also inhibited the serum-stimulated increase in cell number to a maximum of 77% by 1 mumol/L Ang-(1-7). The attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) was unaffected by antagonists selective for angiotensin type 1 (AT1) or type 2 (AT2) receptors; however, [Sar1,Ile1]Ang II and [Sar1,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(1-7) was a result of angiotensin receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 314% at 1 mumol/L Ang II. Ang II also increased the total number of cells (to 145% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT1 antagonist losartan or L-158,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(1-7) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(1-7) appears to be mediated by a novel angiotensin receptor that is not inhibited by AT1 or AT2 receptor antagonists.

Oxidized low density lipoprotein and lysophosphatidylcholine stimulate cell cycle entry in vascular smooth muscle cells. Evidence for release of fibroblast growth factor-2.

We have previously shown that oxidized low density lipoprotein (LDL) but not native LDL stimulated DNA synthesis in cultured smooth muscle cells (SMC) and that alpha-tocopherol (vitamin E) inhibited this proliferative response (Lafont, A., Chai, Y. C., Cornhill, J. F. , Whitlow, P. L., Howe, P. H., and Chisolm, G. M.(1995) J. Clin. Invest. 95, 1018-1025). The moiety of oxidized LDL that stimulates DNA synthesis and the cellular mechanism for this potentially mitogenic effect are not known. We now report that lipid fractions containing lysophospholipids from oxidized LDL or phospholipase A2-treated native LDL stimulated SMC DNA synthesis as did palmitoyl lysophosphatidylcholine (lysoPC). Protein kinase C inhibitors and down-regulation of protein kinase C activity by phorbol ester inhibited oxidized LDL- and lysoPC-induced DNA synthesis. A neutralizing monoclonal antibody against fibroblast growth factor-2 significantly inhibited oxidized LDL and lysoPC-induced DNA synthesis in SMC; irrelevant antibodies were ineffective. Vitamin E inhibited the DNA synthesis stimulated by lysoPC, an observation that distinguished this effect from DNA synthesis induced by another detergent, digitonin. These results suggest that oxidized LDL and its lysoPC moiety stimulate SMC to enter the cell cycle via an oxidative mechanism that causes the release of fibroblast growth factor-2 and a subsequent autocrine or paracrine response.