Steven P. Wrenn

Atherosclerotic Plaque Progression and Vulnerability to Rupture Angiogenesis as a Source of Intraplaque Hemorrhage

Observational studies of necrotic core progression identify intraplaque hemorrhage as a critical factor in atherosclerotic plaque growth and destabilization. The rapid accumulation of erythrocyte membranes causes an abrupt change in plaque substrate characterized by increased free cholesterol within the lipid core and excessive macrophage infiltration. Neoangiogenesis is associated closely with plaque progression, and microvascular incompetence is a likely source of intraplaque hemorrhage. Intimal neovascularization is predominantly thought to arise from the adventitia, where there are a plethora of pre-existing vasa vasorum. In lesions that have early necrotic cores, the majority of vessels invading from the adventitia occur at specific sites of medial wall disruption. A breech in the medial wall likely facilitates the rapid in-growth of microvessels from the adventitia, and exposure to an atherosclerotic environment stimulates abnormal vascular development characterized by disorganized branching and immature endothelial tubes with “leaky” imperfect linings. This network of immature blood vessels is a viable source of intraplaque hemorrhage providing erythrocyte-derived phospholipids and free cholesterol. The rapid change in plaque substrate caused by the excessive accumulation of erythrocytes may promote the transition from a stable to an unstable lesion. This review discusses the potential role of intraplaque vasa vasorum in lesion instability as it relates to plaque rupture.

Bursting Bubbles and Bilayers

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol) (PEG) - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those - not necessarily involving microubbles - to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly “theranostic” vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery.

Measuring raft size as a function of membrane composition in PC-based systems: Part 1--binary systems.

This work applied two steady-state fluorescence techniques to detect nanoscopic membrane domains in a binary dimyristoylphosphocholine (DMPC)-cholesterol system and a ternary dioleoylphosphocholine (DOPC)-dipalmitoylphosphocholine (DPPC)-cholesterol system. A polarity-induced spectral shift in the emission spectra of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) in combination with a Förster resonance energy transfer (FRET) assay agreed with the phase diagrams that have been published for these systems and were observed to be useful tools in the detection of membrane heterogeneities. The DAN-PC/dehydroergosterol (DHE) FRET pair was found to be best suited for use with these steady-state techniques because of their differential partitioning between phases, although a high acceptor concentration was needed to obtain accurate measurements. In the binary system, this high probe concentration was found to be perturbing, but in more representative ternary systems, the high probe concentration no longer disrupted the phase behavior of the system. This FRET pair allowed for the calculation of nanometer-scale domain sizes in model ternary systems, using the two steady-state fluorescence techniques along with a clear and straightforward model.

Determination of microbubble cavitation threshold pressure as function of shell chemistry

AbstractThe sensitivity of inertial cavitation threshold to changes in shell viscosity and elasticity makes shell chemistry (here, polyethylene glycol (PEG) molecular weight and composition) a potential tuning parameter for microbubble based ultrasound contrast and drug delivery applications. It is anticipated that microbubble shell chemistry can be used to adjust the inertial cavitation threshold so as to either avoid or achieve cavitation at a given operating pressure. Here such ideas are tested by measuring the inertial cavitation threshold for populations of phospholipid shelled microbubbles suspended in aqueous media, and this method is used to quantify the influence of shell chemistry on the inertial cavitation threshold. The experimental cavitation data are fitted with a modification of the Herring equation, using shell viscosity and elasticity as the tuning parameters. It is concluded that the design and synthesis of microbubbles with a prescribed inertial cavitation threshold is feasible using PE...

Effect of Sphingomyelinase-Mediated Generation of Ceramide on Aggregation of Low-Density Lipoprotein

This study addresses the response-to-retention hypothesis, which states that the subendothelial retention of atherogenic lipoproteins is the necessary and sufficient condition for the initiation of atherosclerosis. Here we focus on the relationship between the generation of ceramide in the low-density lipoprotein (LDL) phospholipid monolayer and the resulting aggregation of LDL particles. This study provides the first measurement of neutral, Mg (2+)-dependent Sphingomyelinase (Smase)-mediated ceramide formation from LDL-sphingomyelin and does so for a range of enzyme concentrations (0-0.22 units Smase/mL). The kinetics of ceramide generation was measured using a fluorescence assay for the above enzyme concentrations with a fixed substrate concentration (0.33 mg LDL/mL). The kinetics of LDL aggregate formation was measured by dynamic light scattering (DLS, method of cumulants) for identical enzyme concentrations. Ceramide concentration profiles were fit with a modification of the Michaelis-Menten model ( k a = 1.11 x 10 (-1) microM (-1) min (-1), k -a = 6.54 x 10 (2) microM (-1) min (-1), k 1 = 3.33 x 10 (1) microM (-1) min (-1), k -1 = 1.41 x 10 (-2) min (-1), k cat = 8.05 x 10 (1) min (-1), K M = 2.418 microM, k deact = 4.66 x 10 (-2) microM (-1) min (-1)) that accounts for the effects of enzyme attachment to the LDL monolayer and for deactivation of Smase due to product inhibition. LDL aggregation is described by a mass action model as explained in previous studies. A key result of this work is the finding that LDL aggregate size depends directly on ceramide concentration and is independent of enzyme concentration. This study demonstrates how principles of colloid science are relevant to important biomedical problems.

Detection and characterization of laterally phase separated cholesterol domains in model lipid membranes

Abstract We present evidence that laterally phase separated cholesterol domains constitute a new, equilibrium phase in biological membranes. The domains are characterized in multi-lamellar vesicles (MLV) made of cholesterol and dimyristoylphosphatidylcholine (DMPC) but are also shown to exist in biologically relevant, egg lecithin systems containing a mixture of phospholipids. This work utilizes the fluorescent membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesufonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3β-ol (ERGO), which have been shown to be minimally invasive mimics of native membrane lipids. The highlight of the work is a heating-induced alleviation of a DANSYL blue shift at relatively high (but undersaturated) cholesterol loadings, which is reversible through at least three heating and cooling cycles. Comparison of the DANSYL spectral shifts with published DMPC–cholesterol phase diagrams shows unequivocally that the spectral results cannot be explained in terms of previously understood phase behavior. Rather, a lateral phase separation occurs within the vesicle bilayer, giving rise to cholesterol micro-domains. The cholesterol domains appear to coexist with, and should not be confused with, the well-known liquid-order phase that arises because of the cholesterol condensation effect. Additional studies involving ERGO–DANSYL energy transfer show a sequestration of probes within the bilayer, confirming the DANSYL spectral data, and a model that includes domains provides the best description of measured energy transfer efficiencies. Best fits of the energy transfer data, using a mathematical model developed to account for the presence of domains, indicates the domain size to be in the range 10–20 nm.

Influence of shell composition on the resonance frequency of microbubble contrast agents.

The effect of variations in microbubble shell composition on microbubble resonance frequency is revealed through experiment. These variations are achieved by altering the mole fraction and molecular weight of functionalized polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell and measuring the microbubble resonance frequency. The resonance frequency is measured via a chirp pulse and identified as the frequency at which the pressure amplitude loss of the ultrasound wave is the greatest as a result of passing through a population of microbubbles. For the shell compositions used herein, we find that PEG molecular weight has little to no influence on resonance frequency at an overall PEG mole fraction (0.01) corresponding to a mushroom regime and influences the resonance frequency markedly at overall PEG mole fractions (0.050-0.100) corresponding to a brush regime. Specifically, the measured resonance frequency was found to be 8.4, 4.9, 3.3 and 1.4 MHz at PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.075. At an overall PEG mole fraction of just 0.01, on the other hand, resonance frequency exhibited no systematic variation, with values ranging from 5.7 to 4.9 MHz. Experimental results were analyzed using the Sarkar bubble dynamics model. With the dilatational viscosity held constant (10(-8) N·s/m) and the elastic modulus used as a fitting parameter, model fits to the pressure amplitude loss data resulted in elastic modulus values of 2.2, 2.4, 1.6 and 1.8 N/m for PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.010 and 4.2, 1.4, 0.5 and 0.0 N/m, respectively, at an overall PEG mole fraction of 0.075. These results are consistent with theory, which predicts that the elastic modulus is constant in the mushroom regime and decreases with PEG molecular weight to the inverse 3/5 power in the brush regime. Additionally, these results are consistent with inertial cavitation studies, which revealed that increasing PEG molecular weight has little to no effect on inethe rtial cavitation threshold in the mushroom regime, but that increasing PEG molecular weight decreases inertial cavitation markedly in the brush regime. We conclude that the design and synthesis of microbubbles with a prescribed resonance frequency is attainable by tuning PEG composition and molecular weight.

Cholestan‐3β,5α,6β‐triol, but not 7‐ketocholesterol, suppresses taurocholate‐induced mucin secretion by cultured dog gallbladder epithelial cells

In order to investigate oxysterol‐mediated effects on the biliary system, we studied the effects of cholestan‐3β,5α,6β‐triol (TriolC) and 7‐ketocholesterol (7KC) on gallbladder epithelial cells. We compared their cell proliferation effects in cultured dog gallbladder epithelial cells (DGBE) to their effects in cultured human pulmonary artery endothelial cells (HPAE). Oxysterols inhibited cell proliferation in a dose‐dependent fashion. Oxysterols inhibited cell growth to 50% of control at a higher dose for DGBE cells than for HPAE cells. TriolC was more cytotoxic than 7KC. We also investigated the effect of oxysterols on bile salt‐induced mucin secretion by DGBE cells. TriolC suppressed mucin secretion by DGBE cells, whereas 7KC did not. These findings support the hypothesis that biliary oxysterols affect gallbladder mucosal function.

Low-frequency ultrasound-induced transport across non-raft-forming ternary lipid bilayers.

We examined the effect of bilayer composition on membrane sensitivity to low-frequency ultrasound (LFUS) in bilayers composed of ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoyl-phosphocholine (DPPC), and cholesterol. The phase diagram of this system does not display macroscopic phase coexistence between liquid phases (although there are suggestions that there is coexistence between a liquid and a solid phase). Samples from across the composition space were exposed to 20 kHz, continuous wave ultrasound, and the response of the bilayer was quantified using steady-state fluorescence spectroscopy to measure the release of a self-quenching dye, calcein, from large unilamellar vesicles. Dynamic light scattering measurements indicate that, in this system, release proceeds primarily by transport through the vesicle bilayer. While vesicle destruction might account, at least in part, for the light scattering trends observed, evidence of destruction was not as obvious as in other lipid systems. Values for bilayer permeability are obtained by fitting release kinetics to a two-film theory mathematical model. The permeability due to LFUS is found to increase with increasing DPPC content, as the bilayer tends toward the solid-ordered phase. Permeability, and thus sensitivity to LFUS, decreases with either POPC or cholesterol mole fractions. In the liquid regime of this system, there is no recorded phase transition; thus cholesterol is the determining factor in release rates. However, the presence of domain boundaries between distinctly differing phases of liquid and solid is found to cause release rates to more than double. The correlation of permeability with phase behavior might prove useful in designing and developing therapies based on ultrasound and membrane interactions.

Coencapsulation of lipid microbubbles within polymer microcapsules for contrast applications

In this work, the authors examine the acoustic response generated by the coencapsulation of phospholipid shelled microbubbles within the aqueous core of polymer microcapsules and its feasibility as an ultrasound contrast agent. The addition of the polymer shell provides the added benefit of approximately doubling the inertial cavitation threshold of the microbubbles contained within. The feasibility of the utilisation of the coencapsulated contrast agent as a drug delivery vehicle is also discussed. It is concluded that the coencapsulated contrast agent provides contrast similar to that of unencapsulated microbubbles, both in acoustic response and image intensity of contrast to tissue.