Scott M. Colles

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.

Elucidation of a TRPC6-TRPC5 Channel Cascade That Restricts Endothelial Cell Movement

Canonical transient receptor potential (TRPC) channels are opened by classical signal transduction events initiated by receptor activation or depletion of intracellular calcium stores. Here, we report a novel mechanism for opening TRPC channels in which TRPC6 activation initiates a cascade resulting in TRPC5 translocation. When endothelial cells (ECs) are incubated in lysophosphatidylcholine (lysoPC), rapid translocation of TRPC6 initiates calcium influx that results in externalization of TRPC5. Activation of this TRPC6-5 cascade causes a prolonged increase in intracellular calcium concentration ([Ca(2+)](i)) that inhibits EC movement. When TRPC5 is down-regulated with siRNA, the lysoPC-induced rise in [Ca(2+)](i) is shortened and the inhibition of EC migration is lessened. When TRPC6 is down-regulated or EC from TRPC6(-/-) mice are studied, lysoPC has minimal effect on [Ca(2+)](i) and EC migration. In addition, TRPC5 is not externalized in response to lysoPC, supporting the dependence of TRPC5 translocation on the opening of TRPC6 channels. Activation of this novel TRPC channel cascade by lysoPC, resulting in the inhibition of EC migration, could adversely impact on EC healing in atherosclerotic arteries where lysoPC is abundant.

Lysophosphatidylcholine Inhibits Endothelial Cell Migration by Increasing Intracellular Calcium and Activating Calpain

Objective—Endothelial cell (EC) migration, essential for reestablishing arterial integrity after vascular injury, is inhibited by oxidized LDL (oxLDL) and lysophosphatidylcholine (lysoPC) that are present in the arterial wall. We tested the hypothesis that a mechanism responsible for lysoPC-induced inhibition is increased intracellular free calcium concentration ([Ca2+]i). Methods and Results—LysoPC, at concentrations that inhibit in vitro EC migration to 35% of control, increased [Ca2+]i levels 3-fold. These effects of lysoPC were concentration dependent and reversible. LysoPC induced Ca2+ influx within 10 minutes, and [Ca2+]i remained elevated for 2 hours. The calcium ionophore A23187 also increased [Ca2+]i and inhibited EC migration. Chelators of intracellular Ca2+ (BAPTA/AM and EGTA/AM) and nonvoltage-sensitive channel blockers (lanthanum chloride and gadolinium chloride) blunted the lysoPC-induced [Ca2+]i rise and partially preserved EC migration. After lysoPC treatment, calpain, a calcium-dependent cysteine protease, was activated, and cytoskeletal changes occurred. Calpain inhibitors (calpastatin, MDL28170, and calpeptin) added before lysoPC prevented cytoskeletal protein cleavage and preserved EC migration at 60% of control levels. Conclusions—LysoPC increases [Ca2+]i. In turn, activating calpains that can alter the cytoskeleton are activated and EC migration is inhibited.

ISOLATION AND CHARACTERIZATION OF TWO DISTINCT FORMS OF LIVER FATTY ACID BINDING PROTEIN FROM THE RAT

Liver fatty acid binding protein (L-FABP) appears to contain several different forms that may result from post-translational modification or bound ligand. To further assess this possibility, L-FABP was purified from rat liver homogenate and two putative isoforms separated using a sulfonyl column, a strong cation exchange resin. Fraction I eluted at 0.2 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 98% L-FABP. Fraction II eluted at 1.0 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 99% L-FABP. Both fractions contained approx. 0.15 moles of endogenous bound fatty acid per mole of protein, while L-FABP not subjected to the cation exchange step contained 0.75 moles of fatty acid per mole of protein. Fractions I and II had a greater proportion of saturated and monounsaturated fatty acids with a large reduction in polyunsaturated fatty acids compared to L-FABP not fractionated by cation exchange. Mass spectral analysis indicated the molecular mass of Fraction I was 14,315.02 +/- 0.35 Da and Fraction II was 14,315.86 +/- 0.34 Da. The peptide map for each fraction was determined by limited digestion of each fraction with either trypsin, Asp-N, or chymotrypsin to yield overlapping peptide fragments. Mass spectral analysis of these digests indicated the two proteins had identical amino acid fragments and that Cys69 was reduced and there were no Asn to Asp exchanges. Hence, these two forms of L-FABP were not isoforms and were not the result of differences in bound fatty acid. It is proposed that these two distinct forms of rat L-FABP were structural conformers based on two alternative folding pathways.

Protein Kinase Cδ–Dependent Phosphorylation of Syndecan-4 Regulates Cell Migration

Endothelial cell (EC) migration is a complex process requiring exquisitely coordinated focal adhesion assembly and disassembly. Protein kinase C (PKC) is known to regulate focal adhesion formation. Because lysophosphatidylcholine (lysoPC), a major lipid constituent of oxidized low-density lipoprotein, can activate PKC and inhibit EC migration, we explored the signaling cascade responsible for this inhibition. LysoPC increased PKC&dgr; activity, measured by in vitro kinase activity assay, and increased PKC&dgr; phosphorylation. Decreasing PKC&dgr; activation, using pharmacological inhibitors or antisense oligonucleotides, diminished the antimigratory effect of lysoPC. LysoPC-induced PKC&dgr; activation was followed by increased phosphorylation of the transmembrane proteoglycan, syndecan-4, and decreased binding of PKC&agr; to syndecan-4, with a concomitant decrease in PKC&agr; activity. A reciprocal relationship was noted between the interaction of PKC&agr; and &agr;-actinin with syndecan-4. These changes were temporally related to the observed changes in cell morphology and the inhibition of migration of ECs incubated with lysoPC. The data suggested that generalized activation of PKC&dgr; by lysoPC initiated a cascade of events, including phosphorylation of syndecan-4, displacement and decreased activity of PKC&agr;, binding of &agr;-actinin to syndecan-4, and disruption of the time- and site-specific regulation of focal adhesion complex assembly and disassembly required for normal cell migration.

Antioxidant therapy reverses impaired graft healing in hypercholesterolemic rabbits.

OBJECTIVE Limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia contribute to thrombosis and failure of prosthetic grafts. Lipid accumulation and lipid oxidation are associated with decreased EC migration and intimal hyperplasia. The goal of this study was to assess the ability of antioxidants to improve graft healing in hypercholesterolemic animals. METHODS Rabbits were placed in one of four groups: chow plus N-acetylcysteine (NAC), chow plus probucol, chow with 1% cholesterol plus NAC, or chow with 1% cholesterol plus probucol. After 2 weeks, expanded polytetrafluoroethylene grafts (12 cm long x 4-mm internal diameter) were implanted in the abdominal aorta. Grafts were removed after 6 weeks and analyzed for cholesterol content, EC coverage, anastomotic intimal thickness, and the cellular composition of the neointima. Plasma samples were obtained to assess systemic oxidative stress. The data were compared with previously reported data from animals fed diets of chow and chow with 1% cholesterol. RESULTS Prosthetic grafts from rabbits fed chow with 1% cholesterol had significantly greater anastomotic intimal thickening and lower EC coverage than grafts from rabbits fed a regular chow diet. In hypercholesterolemic rabbits, antioxidant therapy decreased global oxidative stress as evidenced by a 40% decrease in plasma thiobarbituric acid reactive substances. In rabbits fed the chow with 1% cholesterol diet, NAC decreased intimal hyperplasia at the proximal anastomosis by 29% and significantly increased graft EC coverage from 46% to 71% (P = .03). Following a similar pattern, probucol decreased intimal hyperplasia by 43% and increased graft EC coverage to 53% in hypercholesterolemic rabbits. CONCLUSIONS Global oxidative stress and anastomotic intimal hyperplasia are increased, and endothelialization of prosthetic grafts is significantly reduced in rabbits fed a high-cholesterol diet. Antioxidant treatment improves EC coverage and decreases intimal hyperplasia. Reducing oxidative stress may promote healing of prosthetic grafts.

Impaired graft healing due to hypercholesterolemia is prevented by dietary supplementation with α-tocopherol

OBJECTIVE The patency of prosthetic grafts is partly limited by incomplete endothelial cell coverage and development of anastomotic intimal hyperplasia. The goal of this study was to determine the effect of elevated cholesterol on prosthetic graft healing and the ability of alpha-tocopherol to improve healing. METHODS Rabbits were placed on one of four diets: chow, chow plus 1% cholesterol, chow plus alpha-tocopherol, or chow plus 1% cholesterol and alpha-tocopherol. After 2 weeks, expanded polytetrafluoroethylene grafts (12-cm long, 4-mm internal diameter) were implanted in the abdominal aorta. Grafts were removed after 6 weeks and analyzed for cholesterol and alpha-tocopherol content, endothelial coverage, anastomotic intimal thickness, and cellular composition of the neointima. RESULTS At the time of graft implantation, plasma cholesterol was 34 +/- 4 mg/dL in the chow group and 689 +/- 30 mg/dL in the 1% cholesterol group (P < .05). Grafts removed from hypercholesterolemic rabbits had marked intimal thickening, with an intima/graft thickness ratio of 0.76 +/- 0.29 compared with 0.14 +/- 0.06 in chow animals (P < .05). Macrophage infiltrate was increased to 45 +/- 11 macrophages/0.625 mm(2) in grafts from hypercholesterolemic rabbits compared with 0 +/- 0.4 in controls (P < .05). Endothelialization of grafts was lower in hypercholesterolemic rabbits than in the chow group, with endothelial cells covering 46% +/- 7% and 62% +/- 7% of the graft surface, respectively (P = .05). When alpha-tocopherol was added to the 1% cholesterol diet, the macrophage count decreased to 12 +/- 8, the intimal/graft thickness ratio decreased to 0.17 +/- 0.09, and endothelial coverage increased to 70% +/- 7% (P < .05 compared with the high-cholesterol group). CONCLUSION Anastomotic intimal hyperplasia is dramatically increased and endothelialization is reduced in rabbits on a high-cholesterol diet, but alpha-tocopherol supplementation blocks the augmented neointimal thickening and improves endothelial cell coverage.

The Effects of Oxidized Lipids and Lipoproteins on Arterial Growth, Remodeling and Restenosis

Studies of restenosis after angioplasty, and more generally studies of arterial responses to injury, have revealed that the variety of potential outcomes includes proliferation of vascular smooth muscle cells, what appears to be a chronic constrictive response, or vascular remodeling. These are not mutually exclusive of one another and they are detailed more fully in other chapters of this book. They are mentioned here to point out that a mechanical arterial injury such as is inflicted by angioplasty, the inflammatory response to that injury and the stimuli for remodeling emanating from flow and pressure alterations caused by the injury, may compete in mediating changes in vascular tone and in the cell migration, proliferation and death that govern the healing process. Thus, basic mechanisms controlling cell death, mitogenesis or vascular relaxation that are mediated by factors already present in atherosclerotic lesions, factors induced by vascular injury or the injury response, and factors elicited by changes in flow or pressure are important to understand. Our purpose in the following pages is to describe a subcategory of the influences on cell growth and vascular reactivity that have been identified in vitro, that is, those resulting from the actions of lipid and lipoprotein oxidation products known to reside in atherosclerotic plaques or enter the angioplasty injury site.