Role of phosphatidylserine in amyloid-beta oligomerization at asymmetric phospholipid bilayers.

Amyloid-beta (Aß1-42) aggregation triggers neurotoxicity and is linked to Alzheimer's disease. Aß1-42 oligomers, rather than extended fibrils, adhere to the cell membrane, causing cell death. Phosphatidylserine (PS), an anionic phospholipid, is prevalent in neuronal membranes (< 20 molar percentage) and, while isolated to the cytoplasmic leaflet of the membrane in healthy cells, its exposure in apoptotic cells and migration to exoplasmic leaflet is triggered by oxidative damage to the membrane. It is widely believed that PS plays a crucial role in the Aß peptide interaction in the membranes of neuronal cells. However, due to the complexity of the cell membrane, it can be challenging to address molecular level understanding of the PS-Aß binding and oligomerization processes. Herein, we use microcavity supported lipid bilayers (MSLBs) to analyse PS and Aß1-42 binding, oligomer formation, and membrane damage. MSLBs are a useful model to evaluate protein-membrane interactions because of their cell-like dual aspect fluidity, their addressability and compositional versatility. We used electrochemical impedance spectroscopy (EIS) and confocal fluorescence microscopy to compare the impact of Aß1-42 on simple zwitterioinic membrane, dioleoylphosphatidylcholine (DOPC), with MSLBs comprised of transversally asymmetric binary DOPC and dioleoylphosphatidylserine (DOPS). Monomeric Aß1-42 adsorbs weakly to the pristine zwitterionic DOPC membrane without aggregation. Using a membrane integrity test, with pyranine trapped within the cavities beneath the membrane, Aß1-42 exposure did not result in pyranine leakage, indicating that DOPC membranes were intact. When 10 mol% DOPS was doped asymmetrically into the membrane's outer leaflet, oligomerization of Aß1-42 monomer was evident in EIS and atomic force microscopy (AFM), and confocal imaging revealed that membrane damage, resulted in extensive pyranine leakage from the pores. The effects were time, and DOPS and Aß1-42 concentration-dependent. Membrane pore formation was visible within 30 minutes, and oligomerization, membrane-oligomer multilayer, and Aß1-42 fibril formation evident over 3 to 18 hours. In asymmetric membranes with DOPS localized to the lower leaflet, optothermally (laser induced) damage increased local DOPS concentrations at the distal leaflet, promoting Aß1-42 aggregation.

Galectin-3 Binding to α5ß1 Integrin in Pore Suspended Biomembranes.

Galectin-3 (Gal3) is a ß-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5ß1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5ß1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane's admittance and fluidity, as well as integrin's lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan-Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5ß1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3-α5ß1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers.

Annexins are soluble membrane-binding proteins that associate in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms crystalline networks that seal or stabilize small membrane tears. Herein, we model this biochemical mechanism to simulate membrane healing at microcavity array supported, transversally asymmetric, lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). Varying annexin V concentration, lipid composition, and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochemical impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was observed over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochemical impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.

A rabbit model for capsular contracture: development and clinical implications.

BACKGROUND: Capsular contracture remains one of the most common complications involving aesthetic and reconstructive breast surgery; however, its cause, prevention, and treatment remain to be fully elucidated. Presently, there is no accurate and reproducible pathologic in vitro or in vivo model examining capsular contracture. The purpose of this study was to establish an effective pathologic capsular contracture animal model that mimics the formation of capsular contracture response in humans. METHODS: New Zealand White rabbits (n = 32) were subdivided into experimental (n = 16) and control groups (n = 16). Each subgroup underwent placement of smooth saline mini implants (30 cc) beneath the panniculus carnosus in the dorsal region of the back. In addition, the experimental group underwent instillation of fibrin glue into the implant pocket as a capsular contracture-inducing agent. Rabbits were euthanized from 2 to 8 weeks after the procedure. Before the animals were euthanized, each implant was serially inflated with saline and a pressure-volume curve was developed using a Stryker device to assess the degree of contracture. Representative capsule samples were collected and histologically examined. Normal and contracted human capsular tissue samples were also collected from patients undergoing breast implant revision and replacement procedures. Tissue samples were assessed histologically. RESULTS: Pressure-volume curves demonstrated a statistically significantly increased intracapsular pressure in the experimental group compared with the control group. The experimental subgroup had thicker, less transparent capsules than the control group. Histologic evaluation of the rabbit capsule was similar to that of the human capsule for the control and experimental subgroups. CONCLUSIONS: The authors conclude that pathologic capsular contracture can be reliably induced in the rabbit. This animal model provides the framework for future investigations testing the effects of various systemic or local agents on reduction of capsular contracture.

Hemodynamics, electrolytes, and organ histology of larger-volume liposuction in a porcine model.

Liposuction is a procedure that allows the surgical removal of excess adipose tissue in healthy individuals. Lipoplasty is commonly performed with few clinical side effects. However, with increased lipoaspirate volumes, complications have been reported. In addition, the abnormal appearance of fat cells in other tissues subsequent to lipoplasty has been reported in a small number of cases. The authors examined whether larger-volume lipoplasty, in the porcine model, resulted in disturbances in cardiac or pulmonary output levels, electrolytes, and liver chemistry analyses or alterations in organ histology. Nine adult porcine specimens were subjected to either lipoplasty (n = 6) with the superwet technique or no lipoplasty (n = 3). Using a Swan-Ganz catheter, cardiac output and pulmonary artery pressure measurements were obtained from initial placement before lipoplasty until 48 hours postoperatively. Blood analyte measurements were obtained. Upon euthanization, liver, kidney, and lung specimens were collected and tissue sections were prepared. No significant differences or trends were observed in cardiac parameters or blood analytes between control and experimental groups. Significant elevations in serum aspartate aminotransferase and alanine aminotransferase enzyme levels (p < 0.03) were observed in animals postoperatively (10 to 48 hours) subjected to lipoplasty compared with controls. Upon gross examination, the lung tissues of animals subjected to lipoplasty unexpectedly demonstrated patchy petechial hemorrhages on the pleural surface. Tissue sections revealed marked hemorrhagic congestion and evidence of pulmonary edema. Fat emboli were also identified within the pulmonary and renal systems.