It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced 125 I-apoA-I binding to the HCBS, whereas 125 IapoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (#187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125 I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-Icontaining particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipidcontaining site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL

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Atherosclerosis is a disease of blood vessel walls that is thought to be initiated as a reaction of insults to the endothelium. The complex sequence of cellular events that begins with focal inflammation leads to the accumulation of leukocytes in the subendothelial layer and unrestricted uptake of oxidized lipoproteins by macrophages and smooth muscle cells, leading to foam cell formation. Vascular endothelial cells do not undergo the foam cell transformation and do not accumulate cholesterol in atherosclerotic plaques to the same extent as macrophages or smooth muscle cells. However, vascular endothelial cells express receptors for oxidized lipoproteins, and have the biochemical pathways for sterol synthesis and receptor-mediated endocytosis of lipoproteins. Data from the authors' laboratory show that high density lipoproteins but not lipid-free apolipoprotein A-I promote cellular cholesterol efflux in human umbilical vascular endothelial cells and human aortic endothelial cells. Gene expression microarrays were used to examine the differential expression of genes after cholesterol loading. While sterol regulatory element-binding protein-sensitive genes were downregulated, the authors identified a novel transporter, the ATP-binding cassette G1 (ABCG1) to be highly expressed in response to both cellular cholesterol loading and stimulation with the liver X receptor agonist 22-hydroxycholesterol. The ABCA1 gene and protein, the major modulator of cellular cholesterol efflux in macrophages and in peripheral and hepatic tissues, are only weakly expressed in human umbilical vascular endothelial cells and human aortic endothelial cells. These data suggest that endothelial cells maintain cholesterol homeostasis by downregulating cholesterol synthesis and low density lipoprotein receptors and by a cellular cholesterol efflux mechanism onto low-affinity but high-capacity high density lipoproteins. The role of ABC-type transporters, including ABCG1, requires further examination.

The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that 125 I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I ⌬(187-243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of 125 I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125 I-apoA-I from the PM was 4-fold slower than that from ICCs at 37°C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL.

HDL is believed to be a potent physiological protective system against atherosclerotic vascular disease. Although it has become generally accepted that this protective effect of HDL is attributable to its pivotal role in the reverse cholesterol transport (RCT) process (1, 2), structural determinants of molecular interactions between circulating HDL particles and key cell proteins governing the RCT process are complex and not well understood.A growing body of evidence indicates that ABCA1 is a critical cell surface protein required for the transfer of cellular lipid and the maintenance of HDL levels in plasma and is likely important for the first step of RCT from peripheral tissues, including macrophages in the vessel wall (3, 4). Furthermore, Brewer and colleagues (5) have documented that hepatic ABCA1 is a key protein for the formation and maintenance of plasma HDL levels. Moreover, the importance of ABCA1 in the lipidation of apolipoprotein A-I (apoA-I) is highlighted by the finding that Ͼ 50 mutations in the ABCA1 gene have been associated with a variety of clinically distinct HDL deficiency diseases, including Tangier disease (TD) and familial HDL deficiency (6, 7). These patients are characterized by extremely low HDL-cholesterol levels, caused by defective transport of cellular cholesterol and phospholipids to the extracellular space, leading to hypercatabolism of lipidpoor nascent HDL particles (8).Earlier studies by Fielding and colleagues (9, 10) have documented that a minor subspecies of human HDL that migrates with pre ␤ mobility on agarose gels can remove free cholesterol from cultured fibroblasts at a faster rate than ␣ -migrating HDL, which constitutes the bulk of plasma HDL. Furthermore, it was documented that pre ␤ -HDL particles were present in the peripheral lymph of dogs (11), suggesting a key role for these particles in the Abbreviations: apoA-I, apolipoprotein A-I; BBSM, bovine brain sphingomyelin; DMPC, dimyristoyl phosphatidylcholine; MLV, multilamellar vesicle; PEG, polyethylene glycol; POPC, palmitoyloleoyl phosphatidylcholine; pre ␤ 1 -LpA-I, pre ␤ 1 apoA-I-containing lipoproteins; RCT, reverse cholesterol transport; SR-BI, scavenger receptor class B type I; TD, Tangier disease; 2D-PAGGE, two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis.

Porous polymers are gaining increased interest in several areas due, in great part, to their large surface area and unique physiochemical properties. Porous polymers are conventionally manufactured using specific processes related to the chemical structure of each polymer. With the wide variety of porous polymers that have been designed, fabricated, and tested to date, this review aims to provide an overview of the advances and recent progress in the preparation processes and fabrication techniques. A detailed comparison between these techniques is also provided. Some of these techniques offer the advantage of controlling the porosity and the possibility to obtain porous 3D polymers. A new generic fabrication process that can be applied to all liquid polymers to texture their outer surfaces with a desired porosity is also presented. The proposed process, which is based on two micromolding steps, offers flexibility in terms of tailoring the texture of the final polymer by simply using porous silicon templates with different pore sizes and configurations. The anticipated process was successfully implemented to texture polyethyl hydrosiloxane (PMHS) using porous silicon and polymethyl methacrylate (PMMA) scaffolds.

Currently, there is no marker in use in the clinical management of colon cancer to predict which patients will respond efficiently to 5‐fluorouracil (5‐FU), a common component of all cytotoxic therapies. Our aim was to develop and validate a multigene signature associated with clinical outcome from 5‐FU therapy and to determine if it could be used to identify patients who might respond better to alternate treatments. Using a panel of 5‐FU resistant and sensitive colon cancer cell lines, we identified 103 differentially expressed genes providing us with a 5‐FU response signature. We refined this signature using a clinically relevant DNA microarray‐based dataset of 359 formalin‐fixed and paraffin‐embedded (FFPE) colon cancer samples. We then validated the final signature in an external independent DNA microarray‐based dataset of 316 stage III FFPE samples from the PETACC‐3 (Pan‐European Trails in Alimentary Tract Cancers) clinical trial. Finally, using a drug sensitivity database of 658 cell lines, we generated a list of drugs that could sensitize 5‐FU resistant patients using our signature. We confirmed using the PETACC‐3 dataset that the overall survival of subjects responding well to 5‐FU did not improve with the addition of irinotecan (FOLFIRI; two‐sided log‐rank test p = 0.795). Conversely, patients who responded poorly to 5‐FU based on our 12‐gene signature were associated with better survival on FOLFIRI therapy (one‐sided log‐rank test p = 0.039). This new multigene signature is readily applicable to FFPE samples and provides a new tool to help manage treatment in stage III colon cancer. It also provides the first evidence that a subgroup of colon cancer patients can respond better to FOLFIRI than 5‐FU treatment alone.

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