Planeación de Operación del Transporte Público

Lipids are transported in the blood as a component o f globular particles called lipoproteins. Lipoproteins also have a role in immune reactions, coagulation and tissue repair (Durrington P.N., 1994). Lipoproteins generally consist of a hydrophobic core of lipids encapsulated by polar phospholipids. Proteins, known as apolipoproteins, are associated with the lipoproteins and serve structural and regulatory functions. The composition of lipoproteins is outlined in figure 1.10 and the metabolic pathway is depicted in figure 1.11.

The LDL of human plasma is composed of a heterogeneous mixture of discrete particle subspecies that differ in their physicochemical and biological properties. LDL isolated from individual subjects is heterogeneous in terms of its density (Nelson C.A. 1977). Plasma LDL has been subfractionated on the basis of density into three major subclasses, namely, light density LDL (1.018-1.030g/ml), intermediate density LDL (1.030-1.040g/ml) and small dense LDL (1.040-1.065g/ml), (Chapman M.J., 1988). Each of the subclasses differs in their physicochemical and biological properties (Lindgren F.T., 1969, Ghosh S., 1993, Nelson C.A., 1977, Rubenstein B., 1978, Shen M.M.S., 1982, Teng B.A., 1985, Patsch W.R., 1982 and Lee D M., 1970).

Hydrodynamic analysis demonstrates that individual LDL subfractions have distinct peak flotation rates and particle sizes, which vary inversely in proportion to density. The flotation rates of subspecies with average densities of 1.0260 to 1.0409 g/ml range from 9.3 to 5.2. Subspecies with average densities of 1.024 to 1.049 g/ml range from 229 to 214 Angstroms in diameter respectively. The molecular weights also decrease progressively with increased density. Individual LDL subclasses typically resolve as unique narrow bands using polyacrylamide gradient gel electrophoresis and each band correlates with a unimodal refractometric profile upon examination by analytical centrifugation.

Examination o f the net electrical charge by agarose gel electrophoresis revealed that each subspecies has discrete mobility and thus unique net negative charge. Intermediate density LDL (1.0297 to 1.0327 g/ml) had the lowest negative charge, while dense LDL (1.0393 to 1.0483g/ml) displayed slightly increased net negative charge.

Analysis o f the composition o f LDL subclasses revealed some common features. Cholesteryl ester is the predominant lipid ester (38.3-42.8%) in all LDL subclasses and tends to diminish as density increases. Triglycerides are a minor component (3-5%). The proportion o f free cholesterol tends to diminish within a narrow range between subclasses (8.5-11.6%). The ratios o f cholesteryl ester to free cholesterol or phospholipid increase with density whereas the free cholesterol to phospholipid ratio is constant. The phospholipid to protein ratio was constant in light density LDL (1.0260 to 1.0314 g/ml) but was reduced 0.2-fold as density increased.

Apolipoprotein B-lOO was the major protein in all subfractions and was slightly more than 1 molecule per LDL particle. Apo BlOO is the sole apoprotein in all subspecies o f LDL from 1.0271-1.0393g/ml, however, Apo E and apo (a) were detected in subspecies o f LDL above and below these densities. Trace amounts o f Apo C lll were detected at densities greater than 1.0358g/ml.

Intermediate density LDL binds with higher affinity to the LDL receptor than either light or dense LDL (Nigon F., 1991). As a consequence, intermediate LDL is degraded in vivo more quickly than the light or dense subspecies o f LDL.

The differences in the physicochemical properties o f human LDL probably arises due to differences in their origins and metabolism and possibly predicts their fate and may further be indicative o f their atherogenicity.

A landmark lipid intervention trial (Steinberg D., 1997a) conclusively showed a cause-and- effect relationship between blood cholesterol levels and risk of coronary heart disease. However, myocardial infarction is evident in people with low cholesterol levels (<200) and patients with high cholesterol levels (>300) may show no clinical signs of CHD (Steinberg D., 1997a). Further studies indicate that it is the oxidation status of LDL that modulates the impact of hypercholesterolaemia on the blood vessel wall.

An association has been established between the titre of antibodies to oxLDL and the rate at which atherosclerosis progresses (Khoo J.C., 1990). The antioxidant, probucol, reduces plasma and aortic wall oxysterol levels in cholesterol-fed rabbits (Hodis H.N., 1992 and Stalenhoef A.F.H., 1993). Results derived from several different animal models using several different antioxidants (probucol, butylated hydroxytoluene, diphenylphenylenediamine and

vitamin E) confirm the role of oxidation in atherogenesis (Carew T.E., 1987 and Kita T., 1987, Steinberg D., 1997a). The efficacy of the compounds in inhibiting LDL from oxidation ex vivo and their ability to inhibit atherogenesis is correlated (Sasahara M., 1994). In addition, patients randomised to 400-800 lU vitamin E showed 47% fewer non-fatal myocardial infarctions and cardiovascular fatalities than the placebo group (Stephens N.G., 1996).

An increase in small dense LDL is associated with increased risk of cardiovascular disease (Austin M.A., 1988, Campos H., 1992 and Chapman M.J., 1998). Several mechanisms have been proposed to account for the increased atherogenicity of small, dense LDL. Penetration of small, dense LDL to the arterial is facilitated by their small particle size (-260 Angstroms). In addition, the subendothelial deposition of small, dense LDL may be exacerbated by their low affinity for the LDL receptor (Nigon F., 1991) and the consequent increase in plasma residence time (Packard C.J., 1997). Proteoglycans in the intimai intracellular matrix selectively bind small dense LDL, sequestering this lipoprotein in a pro-oxidative environment (Anber V., 1997). This may be an important factor contributing to the increased susceptibility of small, dense LDL to oxidative modification (Dejager S., 1993, Tribble D.L. 1992 and de Graaf J., 1991). The increased propensity of small dense LDL to oxidation may also arise from a relative deficit of lipophilic antioxidants (ubiquinol-10, vitamin E and oxygenated carotenoids), (Tribble D.L., 1994 and Goulinet S., 1997), an elevated proportion of polyunsaturated fatty acids (de Graf, 1991) and altered surface lipid monolayer (Tribble D.L., 1992).

The mechanism o f oxidation o f LDL deposited in the artery wall has been the subject of intense research. The ability o f endothelial cell and smooth muscle cells to initiate peroxidation of LDL has been demonstrated in vitro. Cocultures of endothelial and smooth muscle cells convert LDL to mmLDL which then promotes the induction of monocyte chemotactic protein 1 which in turn, results in the transendothelial migration of monocytes (Navab M., 1991). In addition, monocytes and leukocytes oxidise LDL and render it cytotoxic (Cathcart M.K., 1985). Modification o f LDL by endothelial cells involves lipid peroxidation and degradation o f phospholipids (Steinbrecher U.P., 1984). Endothelial cell lipoxygenase has been implicated in the oxidative modification o f LDL (Parthasarathay S., 1989). The nature o f the initiating radical is discussed further in section 6 .1.2.

Figure 1.9: Growth regulatory molecules in atherosclerosis

Growth regulator

Source Target and outcome

bFGF Macrophages,

endothelial cells, smooth muscle cells

Endothelial and smooth muscle cell proliferation, endothelial chemotaxis

GM-CSF Endothelial cells Monocyte chemotaxis, proliferation and differentiation

hb-EGF Endothelial cells Smooth muscle cell proliferation IGF Fibroblasts Smooth muscle cell proliferation IL-1 Macrophages,

endothelial cells, smooth muscle cells

Smooth muscle cell proliferation

MCP-1 Endothelial cells Monocyte chemotaxis and transendothelial migration

M-CSF Endothelial cells Monocyte chemotaxis, proliferation and differentiation

PD-ECGF Platelets Proliferation of endothelial cells

PDGF Endothelial cells Smooth muscle cell proliferation and chemotaxis TGFa Macrophages, platelets Endothelial cell proliferation

TGFP Platelets, endothelial cells, macrophages, smooth muscle cells

Smooth muscle cell proliferation. Inhibition of smooth muscle cell proliferation and increased deposition o f matrix (fibronectin, collagen and glycosaminoglycans)

TN Fa Macrophages,

endothelial cells, smooth muscle cells

Smooth muscle cell proliferation

VEGF Macrophages, smooth muscle cells

Endothelial cell proliferation

Figure 1.10: The composition o f plasma lipoproteins

Chylo­

microns VLDL LDL IDL HDL2 HDLa

% of total lipoprotein Protein 1-2 10 18 25 40 55 Lipid 98-99 90 82 75 60 45 % o f total lipids Triglycerides 88 56 32 7 6 7 Cholesterol 3 17 41 59 43 38 Phospholipid 9 19 27 28 42 41 % o f cholesterol esterified 46 57 66 70 74 81

Figure 1.11: Antioxidant and lipid composition of LDL mol/mol LDL Fatty acids Palmitic acid 693 Palmitoleic acid 44 Stearic acid 143 Oleic acid 454 Linoleic acid 1100 Arachidonic acid 153 Docosahexanoic acid 29 Antioxidants a-tocopherol 6.37 y-tocopherol 0.51 p-carotene 0.29 a-carotene 0.12 Lycopene 0.16 Cryptoxanthin 0.14 Cantaxanthin 0.02 Lutein + zeaxanthin 0.04 Phytofluene 0.05 Ubiquinol-10 0.10

Total PUP As (mean) 1283

Total antioxidants (mean) 7.8

Hç-patocyte T G & C E VLDL LCAT T G & C E Chvlomicron T G & C E P enphefaJ c e il

mucosal:

ceil

Legend: A, B, C and E refer to the corresponding apoproteins and FC, PL, CE and TG to free cholesterol, phospholipid, cholesterol ester and triglyceride respectively. Rem; chylomicron remnants, HDL; high density lipoprotein, VLDL; very low density lipoprotein, LDL; low density lipoprotein, LPL; lipoprotein lipase, HPL; hepatic lipase, LCAT; lecithin: cholesterol acyl transferase

Oxidised LDL represents a broad spectrum of modified particles that range from mildly (mmLDL) to extensively modified (oxLDL). Highly oxidised LDL (oxLDL) contains abundant lipid peroxidation products (Brown M.S., 1983) including aldehydes. The formation of aldehydes results in the loss of amino groups from apo BlOO, as a consequence oxLDL has increased electrophoretic mobility (Esterbauer H., 1992) and is recognised by the scavenger receptor (Parthasarathy S., 1986 and Henriksen T., 1983).

Minimally-modified LDL is LDL that has undergone antioxidant depletion and oxidation of arachidonate-containing phospholipid but much less linoleic oxidation. MmLDL has little or no protein modification and is taken up by the native LDL receptor (Berliner J.A., 1990 and Watson A. D,, 1995). In addition, mmLDL induces differentiation factors (Rajavashisth T.B., 1990) as well as the transendothelial migration of monocytes via the induction of monocyte chemotactic protein-1 (M CPl) in smooth muscle and endothelial cells (Berliner J.A., 1990, Navab M., 1988 and Navab M., 1991).

Minimal modification of LDL is insufficient to alter its interaction with the LDL receptor or to make it a ligand for the acetyl LDL receptor (Berliner J.A., 1990). The impact of LDL oxidation upon the progression of atherosclerosis is in part due to the ability of mmLDL and oxLDL to induce a number of proatherogenic responses by arterial cells (figure 1.8).

The proatherogenic responses of arterial cells to the various LDL species include the induction of; increased permeability in endothelial cells, adhesion molecules, chemotactic factors, growth factors, haemostatic factors and cytotoxicity. Instigation of these responses contributes to the progression of atherosclerosis by enhancing the formation of subendothelial deposits of foam cells due to transendothelial migration, proliferation and differentiation of monocytes, lymphocytes and neutrophils. In addition, intimai hyperplasia results from the ability of oxidised LDL to promote migration and proliferation of smooth muscle cells. A prothrombotic status may result from the activation of platelet aggregation, the enhancement of thrombosis and reduction of fibrinolysis. Plaque instability results from an increase in the lipid: collagen ratio (Davies M.J., 1993). Oxidised LDL also contributes to plaque instability due to its cytotoxic effects upon smooth muscle cells and foam cells, which result in a reduction in collagen composition and increased lipid content of plaques, respectively.

Importantly, the studies tabulated in figure 1.8 also demonstrate that native, non-oxidised LDL induces proatherogenic responses including; the induction of the monocyte chemotactic factor MCP-1, endothelial adhesion molecules ICAM-1, VCAM-1, E-selectin and the cytokine PDGF and expression o f the scavenger receptor CD36. Induction of these factors facilitates the progression of atherosclerosis as a result of the promotion of the adhesion and

transendothelial migration of monocytes and T-cells and their transformation to foam cells as well as the induction of smooth muscle cell proliferation.

Thus, both native and oxidised LDL are capable of promoting atherosclerosis, however, oxLDL is more precisely correlated with the risk of cardiovascular disease (Khoo J.C., 1990), This may arise from a differential in the response of cells to native versus oxidised LDL, for instance oxidised and not native LDL results in increased permeability in endothelial monolayers. Increased endothelial permeability is a critical factor in the initiation stages of atherosclerosis since it permits the deposition of lipid in the subendothelial space. Some studies have addressed the issue of differential responses to native and oxidised LDL, (although many have not), and this subject is discussed further in section 6.6.1.