Nassrin Dashti

Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy

The principal extracellular lesions of age-related maculopathy (ARM), the leading cause of vision loss in the elderly, involve Bruchs membrane (BrM), a thin vascular intima between the retinal pigment epithelium (RPE) and its blood supply. With age, 80–100 nm solid particles containing esterified cholesterol (EC) accumulate in normal BrM, and apolipoprotein B (apoB) immunoreactivity is detectable in BrM- and ARM-associated lesions. Yet little evidence indicates that increased plasma cholesterol is a risk factor for ARM. To determine if RPE is capable of assembling its own apoB-containing lipoprotein, we examined RPE for the expression of microsomal triglyceride transfer protein (MTP), which is required for this process. Consistent with previous evidence for apoB expression, MTP is expressed in RPE, the ARPE-19 cell line, and, unexpectedly, retinal ganglion cells, which are neurons of the central nervous system. De novo synthesis and secretion of neutral lipid by ARPE-19 was supported by high levels of radiolabeled EC and triglyceride in medium after supplementation with oleate. Lipoprotein assembly and secretion is implicated as a constitutive retinal function and a plausible candidate mechanism involved in forming extracellular cholesterol-containing lesions in ARM. The pigmentary retinopathy and neuropathy of abetalipoproteinemia (Mendelian Inheritance of Man 200100; Bassen-Kornzwieg disease), which is caused by mutations in the MTP gene, may involve loss of function at the retina.

Plasma apolipoproteins and risk for age related maculopathy

Aim: To determine if elevated plasma levels of atherogenic and/or anti-atherogenic lipoproteins are risk factors for developing age related maculopathy (ARM). Methods: In a cross sectional study in a university clinic setting, 129 patients (72 women and 57 men) underwent colour fundus photography, acuity and contrast sensitivity assessment, and electroimmunoassays of plasma apolipoproteins B (apoB) and A-I (apoA-I), the principal proteins of low density and high density lipoproteins, respectively. Maculopathy stage was assigned using the AREDS grading system. Results: Levels of apoB in no ARM, mild, intermediate, and advanced ARM groups were 93.3, 91.8, 95.2, and 98.2 mg/dl, respectively. Levels of apoA-I were 147.4, 148.6, 141.0, and 144.9 mg/dl in the same groups. There was no significant association between these measures, typical for age, and maculopathy stage. Conclusion: Although drusen associated with ARM and ageing contain cholesterol and apoB, like the lipid rich core of an atherosclerotic plaque, the results of this study and our previous work in toto make the prospects of a plasma origin for these lesion constituents increasingly untenable. This conclusion is consistent with an emerging hypothesis that a large lipoprotein of intraocular origin is an important pathway for constituent retinal lipid processing and the biogenesis of drusen.

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells. This approach led to 98% reduction in MTP protein levels in both cell types. Metabolic labeling studies demonstrated a drastic 90–95% decrease in the secretion of rat endogenous apoB100-containing lipoproteins in MTP-deficient McA-RH7777 cells compared with cells transfected with negative control miR-shRNA. A similar reduction was observed in the secretion of rat endogenous apoB48 under the experimental conditions employed. In contrast, MTP absence had no significant effect on the synthesis, lipidation, and secretion of human apoB:1000-containing particles. These results provide strong evidence in support of the concept that in McA-RH7777 cells, acquisition of PL by apoB:1000 and initiation of apoB-containing lipoprotein assembly, a process distinct from the conventional first-step assembly of HDL-sized apoB-containing particles, do not require MTP. This study indicates that, in hepatocytes, a factor(s) other than MTP mediates the formation of the PL-rich primordial apoB:1000-containing initiation complex.

Relationship between Amphipathic β Structures in the β1 Domain of Apolipoprotein B and the Properties of the Secreted Lipoprotein Particles in McA-RH7777 Cells

Our previous studies demonstrated that the first 1000 amino acid residues (the βα1 domain) of human apolipoprotein (apo) B-100, termed apoB:1000, are required for the initiation of lipoprotein assembly and the formation of a monodisperse stable phospholipid (PL)-rich particle. The objectives of this study were (a) to assess the effects on the properties of apoB truncates undergoing sequential inclusion of the amphipathic β strands in the 700 N-terminal residues of the β1 domain of apoB-100 and (b) to identify the subdomain in the β1 domain that is required for the formation of a microsomal triglyceride transfer protein (MTP)-dependent triacylglycerol (TAG)-rich apoB-containing particle. Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. (1) The presence of amphipathic β strands in the 200 N-terminal residues of the β1 domain resulted in the secretion of apoB truncates (apoB:1050 to apoB:1200) as both lipidated and lipid-poor particles. (2) Inclusion of residues 300-700 of the β1 domain led to the secretion of apoB:1300, apoB:1400, apoB:1500, and apoB:1700 predominantly as lipidated particles. (3) Particles containing residues 1050-1500 were all rich in PL. (4) There was a marked increase in the lipid loading capacity and TAG content of apoB:1700-containing particles. (5) Only the level of secretion of apoB:1700 was markedly diminished by MTP inhibitor BMS-197636. These results suggest that apoB:1700 marks the threshold for the formation of a TAG-rich particle and support the concept that MTP participates in apoB assembly and secretion at the stage where particles undergo a transition from PL-rich to TAG-rich.