High density lipoprotein (HDL) associated paraoxonase-1 (PON1) is crucial for the anti-oxidant, anti-inflammatory, and anti-atherogenic properties of HDL. Discoidal apolipoprotein (apo)A-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) complex has been shown to be the most effective in binding PON1, stabilizing it, and enhancing its lactonase and inhibitory activity of low density lipoprotein oxidation. Based on our earlier study demonstrating that apoA-I mimetic peptide 4F forms discoidal complex with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, we hypothesized that lipid complexes of 4F would be able to bind PON1 and enhance its activity and stability. To test our hypothesis, we have expressed and purified a recombinant PON1 (rPON1) and studied its interaction with 4F:POPC complex. Our studies show significant increase, compared to the control, in the paraoxonase activity and stability of rPON1 in the presence of 4F:POPC complex. We propose that 4F:POPC complex is a novel platform for PON1 binding, increasing its stability, and enhancing its enzyme activity. We propose a structural model for the 4F:POPC:PON1 ternary complex that is consistent with our results and published observations.
High density lipoprotein; Peptide; Amphipathic helix; Apolipoprotein A-I; Lipid; Paraoxonase-1
Framingham Risk Scores (FRS) were validated in a mostly Caucasian population. Evaluation of subclinical atherosclerosis by carotid ultrasound may improve ascertainment of risk in non-White populations. This study aimed to evaluate carotid intima-media thickness (cIMT) and carotid plaquing among Mexican Americans, and to correlate these markers with coronary risk factors and the FRS.
Participants (n=141) were drawn from the Cameron County Hispanic Cohort. Carotid artery ultrasound was performed and cIMT measured. Carotid plaque was defined as areas of thickening >50% of the thickness of the surrounding walls. Mean age was 53.1±11.7 years (73.8% female). Most were overweight or obese (88.7%) and more than half (53.2%) had the metabolic syndrome. One third (34.8%) had abnormal carotid ultrasound findings (either cIMT ≥75th percentile for gender and age or presence of plaque). Among those with abnormal carotid ultrasound, the majority were classified as being at low 10-year risk for cardiovascular events. Carotid ultrasound reclassified nearly a third of the cohort as being at high risk. This discordance between 10-year FRS and carotid ultrasound was noted whether risk was assessed for hard coronary events or global risk. Concordance between FRS and carotid ultrasound findings was best when long-term (30-year) risk was assessed and no subject with an abnormal carotid ultrasound was categorized as low risk by the 30-year FRS algorithm.
Integration of carotid ultrasound findings to coronary risk assessments and use of longer term prediction models may provide better risk assessment in this minority population, with earlier initiation of appropriate therapies.
Carotid ultrasound; Subclinical atherosclerosis; Framingham risk score; Minority population; Risk assessment
Delayed cerebral vasospasm following subarachnoid hemorrhage causes severe ischemic neurologic deficits leading to permanent neurologic dysfunction or death. Reduced intravascular and perivascular nitric oxide (NO) availability is a primary pathophysiology of cerebral vasospasm. In this study, we evaluated NO-loaded echogenic liposomes (NO-ELIP) for ultrasound-facilitated NO delivery to produce vasodilation for treatment of vasospasm following subarachnoid hemorrhage. We investigated the vasodilative effects of NO released from NO-ELIP both ex vivo and in vivo. Liposomes containing phospholipids and cholesterol were prepared, and NO was encapsulated. The encapsulation and release of NO from NO-ELIP were determined by the syringe/vacuum method and ultrasound imaging. The ex vivo vasodilative effect of NO-ELIP was investigated using rabbit carotid arteries. Arterial vasodilation was clearly observed with NO-ELIP exposed to Doppler ultrasound whereas there was little vasodilative effect without exposure to Doppler ultrasound in the presence of red blood cells. Penetration of NO into the arterial wall was determined by fluorescent microscopy. The vasodilative effects of intravenously administered NO-ELIP in vivo were determined in a rat subarachnoid hemorrhage model. NO-ELIP with ultrasound activation over the carotid artery demonstrated effective arterial vasodilation in vivo resulting in improved neurologic function. This novel methodology for ultrasound-controlled delivery of NO has the potential for therapeutic treatment of vasospasm following subarachnoid hemorrhage. This ultrasound-controlled release strategy provides a new avenue for targeted bioactive gas and therapeutic delivery for improved stroke treatment.
vasospasm; vasodilation; subarachnoid hemorrhage; nitric oxide; liposomes; ultrasound
A novel method for the facile production of gas-containing liposomes with simultaneous drug encapsulation is described. Liposomes of phospholipid and cholesterol were prepared by conventional procedures of hydrating the lipid film, sonicating, freezing and thawing. A single, but critical modification of this procedure generates liposomes that contain gas (air, perfluorocarbon, argon); after sonication, the lipid is placed under pressure with the gas of interest. After equilibration, the sample is frozen. The pressure is then reduced to atmospheric and the suspension thawed. This procedure leads to entrapment of air in amounts up to 10% by volume by lipid dispersions at moderate (10mg/ml) concentrations of lipids. The amount of gas encapsulated increases with gas pressure and lipid concentration. Utilizing 0.32 M mannitol to provide an aqueous phase with physiological osmolarity, 1, 3, 6 or 9 atm of pressure was applied to 4 mg of lipid. This led to encapsulation of 10, 15, 20, and 30 μl of gas in a total of 400 μl of liposome dispersion (10mg lipids/ml), respectively. The mechanism for gas encapsulation presumably depends upon the fact that air (predominantly nitrogen and oxygen), like most solutes, dissolves poorly in ice and is excluded from the ice that forms during freezing. The excluded air then comes out of solution as air pockets that are stabilized in some form by a lipid coating. The presence of air in these preparations sensitizes them to ultrasound (1MHz, 8 W/cm2,10 second) such that up to half of their aqueous contents (which could be a water soluble drug) can be released by short (10 s) applications of ultrasound. Both diagnostic and therapeutic applications of the method are conceivable.
Phospholipid freeze-thawing; controlled release; drug encapsulation; ultrasound
Ultrasound (US)-enhanced thrombolytic treatment protocols currently in clinical trials for stroke applications involve systemic administration of tissue plasminogen activator (tPA; Alteplase), which carries a risk of adverse bleeding events. The present study aimed to compare the thrombolytic efficacy of a tPA-loaded echogenic liposome (ELIP) formulation with insonification protocols causing rapid fragmentation or acoustically-driven diffusion.
Materials and Methods
Thrombi were induced in the abdominal aortas of male New Zealand white rabbits (2–3 kg) using thrombin and a sclerosing agent (sodium ricinoleate) after aortic denudation with a balloon catheter. Thrombolytic and cavitation nucleation agents (200 μg of tPA alone, tPA mixed with 50 μg of a microbubble contrast agent, or tPA-loaded ELIP) were bolus-injected proximal to the clot through a catheter introduced into the abdominal aorta from the carotid artery. Clots were exposed to transabdominal color Doppler US (6 MHz) for 30 minutes at a low mechanical index (MI = 0.2) to induce sustained bubble activity (acoustically-driven diffusion), or for 2 minutes at an MI of 0.4 to cause ELIP fragmentation. Degree of recanalization was determined by Doppler flow measurements distal to the clots.
All treatments showed thrombolysis, but tPA-loaded ELIP was the most efficacious regimen. Both US treatment strategies enhanced thrombolytic activity over control conditions.
The thrombolytic efficacy of tPA-loaded ELIP is comparable to other clinically described effective treatment protocols, while offering the advantages of US monitoring and enhanced thrombolysis from a site-specific delivery agent.
thrombolysis; ultrasound; plasminogen activators; thrombus
Intravascular ultrasound (IVUS) is a standard imaging modality for identification of plaque formation in the coronary and peripheral arteries. Volumetric three-dimensional (3D) IVUS visualization provides a powerful tool to overcome the limited comprehensive information of 2D IVUS in terms of complex spatial distribution of arterial morphology and acoustic backscatter information. Conventional 3D IVUS techniques provide sub-optimal visualization of arterial morphology or lack acoustic information concerning arterial structure due in part to low quality of image data and the use of pixel-based IVUS image reconstruction algorithms. In the present study, we describe a novel volumetric 3D IVUS reconstruction algorithm to utilize IVUS signal data and a shape-based nonlinear interpolation.
We developed an algorithm to convert a series of IVUS signal data into a fully volumetric 3D visualization. Intermediary slices between original 2D IVUS slices were generated utilizing the natural cubic spline interpolation to consider the nonlinearity of both vascular structure geometry and acoustic backscatter in the arterial wall. We evaluated differences in image quality between the conventional pixel-based interpolation and the shape-based nonlinear interpolation methods using both virtual vascular phantom data and in vivo IVUS data of a porcine femoral artery. Volumetric 3D IVUS images of the arterial segment reconstructed using the two interpolation methods were compared.
In vitro validation and in vivo comparative studies with the conventional pixel-based interpolation method demonstrated more robustness of the shape-based nonlinear interpolation algorithm in determining intermediary 2D IVUS slices. Our shape-based nonlinear interpolation demonstrated improved volumetric 3D visualization of the in vivo arterial structure and more realistic acoustic backscatter distribution compared to the conventional pixel-based interpolation method.
This novel 3D IVUS visualization strategy has the potential to improve ultrasound imaging of vascular structure information, particularly atheroma determination. Improved volumetric 3D visualization with accurate acoustic backscatter information can help with ultrasound molecular imaging of atheroma component distribution.
Intravascular ultrasound; Shape-based interpolation; Three-dimensional reconstruction
Resting ischemic electrocardiographic abnormalities have been associated with cardiovascular mortality. Simple markers of abnormal autonomic tone have also been associated with diabetes, obesity, and the metabolic syndrome in some populations. Data on these electrocardiographic abnormalities and correlations with coronary risk factors are lacking among Mexican Americans wherein these conditions are prevalent.
This study aimed to evaluate the prevalent resting electrocardiographic abnormalities among community-dwelling Mexican Americans, and correlate these findings with coronary risk factors, particularly diabetes, obesity, and the metabolic syndrome.
Study subjects (n=1280) were drawn from the Cameron County Hispanic Cohort comprised of community-dwelling Mexican Americans living in Brownsville, Texas at the United States-Mexico border. Ischemic electrocardiographic abnormalities were defined as presence of ST/T wave abnormalities suggestive of ischemia, abnormal Q waves, and left bundle branch block. Parameters that reflect autonomic tone, such as heart rate-corrected QT interval and resting heart rate, were also measured.
Ischemic electrocardiographic abnormalities were more prevalent among older persons and those with hypertension, diabetes, obesity, and the metabolic syndrome. Subjects in the highest quartiles of QTc interval and resting heart rate were also more likely to be diabetic, hypertensive, obese, or have the metabolic syndrome.
Among Mexican Americans, persons with diabetes, obesity, and the metabolic syndrome were more likely to have ischemic electrocardiographic abnormalities, longer QTc intervals, and higher resting heart rates. A resting electrocardiogram can play a complementary role in the comprehensive evaluation of cardiovascular risk in this minority population.
Electrocardiogram; Coronary Risk Factors; Minority; Health Disparity
Melanocortin-3 receptor (MC3R), expressed in the hypothalamus and limbic systems of the brain, as well as by peripheral sites, plays an important role in the regulation of energy homeostasis and other physiological functions. Past work shows that MC3R-deficiency resulted in fat mass increase, feeding efficiency increase, hyperleptinemia and mild hyperinsulinemia in mice and human. MC3R belongs to G-protein coupled receptor (GPCR) family and many studies indicate that some cysteine residues in GPCR play key roles in maintaining receptor tertiary structure and function. In this study, we examined the role of cysteine residues in MC3R on receptor function. Human MC3R (hMC3R) has eighteen cysteine residues where they are located in the extracellular loops (ELs), the transmembrane domains (TMs) and the intracellular loops (ILs). We replaced these cysteines with serine and expressed these receptors in HEK-293 cells which lack endogenous MC3R. Our results indicate that five cysteines in eighteen of the hMC3R are important for hMC3R function. Mutations, C305S, C311S, and C313S in EL3, resulted in significant decrease in receptor expression and receptor function while two other mutations C115S and C162S in TM3 significantly decreased NDP-MSH binding affinity and potency. These results suggest that extracellular cysteine residue 305, 311 and 313 are crucial for receptor expression and the transmembrane cysteine residue, C115 and 162 are important for ligand binding and signaling. These findings provide important insights into the importance of cysteine residues of hMC3R on receptor tertiary structure and function.
MSH; MC3R; cysteine; MCR; GPCR
A pilot study of adults who had onset of juvenile dermatomyositis (JDM) in childhood, before current therapeutic approaches, to characterize JDM symptoms and subclinical cardiovascular disease.
Eight adults who had JDM assessed for disease activity and 8 healthy adults (cardiovascular disease controls) were tested for carotid intima media thickness and brachial arterial reactivity. Adults who had JDM and 16 age-, sex-, and body mass index-matched healthy metabolic controls were evaluated for body composition, blood pressure, fasting glucose, lipids, insulin resistance, leptin, adiponectin, proinflammatory oxidized high-density lipoprotein (HDL), and nail-fold capillary end row loops.
Adults with a history of JDM, median age 38 years (24–44 years) enrolled a median 29 years (9–38 years) after disease onset, had elevated disease activity scores, skin (7/8), muscle (4/8), and creatine phosphokinase (2/8). Compared with cardiovascular disease controls, adults who had JDM were younger, had lower body mass index and HDL cholesterol (P = .002), and increased intima media thickness (P = .015) and their brachial arterial reactivity suggested impairment of endothelial cell function. Compared with metabolic controls, adults who had JDM had higher systolic and diastolic blood pressure, P = .048, P = .002, respectively; lower adiponectin (P = .03); less upper arm fat (P = .008); HDL associated with end row loops loss (r = −0.838, P = .009); and increased proinflammatory oxidized HDL (P = .0037).
Adults who had JDM, 29 years after disease onset, had progressive disease and increased cardiovascular risk factors.
Ultrasound enhances thrombolysis when combined with a thrombolytic and a contrast agent. This study aimed to evaluate the thrombolytic effect of our tissue plasminogen activator (tPA)–loaded echogenic liposomes (ELIP) in an in vivo clot model, with and without ultrasound treatment.
Methods and Results
The femoral arteries of New Zealand White rabbits (n=4 per group) were cannulated. The abdominal aortas were denuded, and thrombi were created using a solution of sodium ricinoleate plus thrombin. Rabbits were then randomly selected to receive tPA-loaded ELIP (200 μg of tPA/5 mg of lipid) or empty ELIP with or without pulsed (color) Doppler ultrasound (5.7 MHz) for 2 minutes. Thrombus was imaged and echogenicity analyzed before and after ELIP injection. Blood flow velocities were measured at baseline, after clot formation, and serially after treatment up to 15 minutes. tPA-loaded ELIP highlighted thrombus in the abdominal aorta more effectively than empty ELIP (P<0.05). Ultrasound enhanced the thrombolytic effect of tPA-loaded ELIP, resulting in earlier and more complete recanalization rates (P<0.001).
This study demonstrates effective highlighting of clots and thrombolytic effect of tPA-loaded ELIP in an in vivo rabbit aorta clot model. Doppler ultrasound treatment enhances this thrombolytic effect, resulting in earlier and more complete recanalization rates.
arterial thrombosis; contrast echo; Doppler ultrasound; echocardiography; thrombolysis
The role of strength training in peripheral arterial disease (PAD) is unclear. Benefits of supervised treadmill exercise in PAD patients without intermittent claudication (IC) are not established.
To determine whether supervised treadmill exercise and lower extremity resistance training, respectively, improve functional performance compared to a control group in PAD persons with and without IC.
Randomized controlled clinical trial performed between 4/1/04 and 8/19/08.
156 people with PAD (ankle brachial index ≤ 0.95), including 81.4% without IC.
Primary outcomes were six-minute walk performance and the short physical performance battery (SPPB). Additional outcomes were brachial artery flow-mediated dilation (FMD), treadmill walking performance, the Walking Impairment Questionnaire (WIQ), and the Short-Form 36 Physical Functioning score (SF-36 PF).
Three parallel arms: supervised treadmill exercise, supervised lower extremity resistance training, and a control group.
Compared to control, the treadmill exercise group increased six-minute walk distance (+35.9 meters, 95% confidence interval (CI), +15.3 to +56.5; P <0.001), while the resistance trained group did not improve (+12.4 meters, 95% CI, −8.42 to +33.3; P=0.24). Neither exercise group improved the SPPB. Compared to control, treadmill exercise improved brachial artery FMD (+1.53%, 95% CI, +0.35 to +2.70, P=0.018), time on treadmill (+3.44 minutes, 95% CI, +2.05 to +4.84; P<0.001), the WIQ distance score P=0.015), and the SF-36 PF score (P=0.02). Compared to control, resistance training improved time on treadmill (+1.98 minutes, 95% CI, +0.56 to +3.39; P=0.007), the WIQ distance score (P=0.02), the WIQ stair climbing score (P=0.02), and the SF-36 PF score (P=0.04).
Supervised treadmill exercise improved six-minute walk distance, treadmill walking performance, brachial artery FMD, and quality of life, but not the SPPB, in PAD participants with and without classic IC symptoms. Resistance training improved treadmill walking performance, quality of life, and stair climbing ability in patients with PAD.
Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and glucose homeostasis. Synthetic nonpeptide compound N- (3R)-1 4-tetrahydroisoquinolinium-3-ylcarbonyl -(1R)-1-(4-chlorobenzyl)-2- 4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl -2-oxoethylamine (THIQ) is a potent agonist at MC4R but not at hMC2R. In this study, we utilized two approaches (chimeric receptor and site-directed mutagenesis) to narrow down the key amino acid residues of MC4R responsible for THIQ binding and signaling. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TMs) of the human MC4R (hMC4R) with the homologous regions of hMC2R were constructed. Our results indicate that the cassette substitutions of these TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter THIQ binding affinity and potency except the substitution of the hMC4R TM3, suggesting that the conserved amino acid residues in these TMs of the hMC4R are main potential candidates for THIQ binding and signaling while non conserved residues in TM3 of MC4R may also be involved. Nineteen MC4R mutants were then created, including 13 conserved amino acid residues and 6 non-conserved amino acid residues. Our results indicate that seven conserved residue [E100 (TM2), D122 (TM3), D126 (TM3), F254 (TM6), W258 (TM6), F261 (TM6), H264 (TM6)] are important for THIQ binding and three non-conserved residues [N123 (TM3), I129 (TM3) and S131 (TM3)] are involved in THIQ selectivity. In conclusion, our results suggest that THIQ utilize both conserved and non-conserved amino acid residues for binding and signaling at hMC4R and non conserved residues may be responsible for MC4R selectivity.
Ischemia-related neurologic injury is a primary cause of stroke disability. Studies have demonstrated that xenon (Xe) may have potential as an effective and nontoxic neuroprotectant. Xe delivery is, however, hampered by lack of suitable administration methods. We have developed a pressurization-freeze method to encapsulate Xe into echogenic liposomes (Xe-ELIP) and have modulated local gas release with transvascular ultrasound exposure.
Methods and Results
Fifteen microliters of Xe were encapsulated into each 1 mg of liposomes (70% Xe and 30% argon). Xe delivery from Xe-ELIP into cells and consequent neuroprotective effects were evaluated with oxygen-glucose deprived and control neuronal cells in vitro. Xe-ELIP were administered into Sprague-Dawley rats intravenously or intraarterially following right middle cerebral artery occlusion. 1-MHz low-amplitude (0.18 MPa) continuous wave ultrasound directed onto the internal carotid artery triggered Xe release from circulating Xe-ELIP. Effects of Xe delivery on ischemia-induced neurologic injury and disability were evaluated. Xe-ELIP delivery to oxygen-glucose deprived neuronal cells improved cell viability in vitro and 48% infarct volume decrease in vivo. Intravenous Xe-ELIP administration in combination with the ultrasound directed onto the carotid artery enhanced local Xe release from circulating Xe-ELIP and demonstrated 75% infarct volume reduction. This was comparable to the effect following intraarterial administration. Behavioral tests on limb placement and grid and beam walking correlated with infarct reduction.
This novel methodology may provide a noninvasive strategy for ultrasound-enhanced local therapeutic gas delivery for cerebral ischemia-related injury while minimizing systemic side effects.
Cerebral ischemia; Contrast media; Stroke; Xenon; Liposomes
This study aimed to demonstrate three-dimensional (3D) visualization of early/inflammatory arterial atheroma using intravascular ultrasound (IVUS) and targeted echogenic immunoliposomes (ELIP). IVUS can be used as a molecular imaging modality with the use of targeted contrast agents for atheroma detection. Three-dimensional reconstruction of 2-dimensional IVUS images may provide improved atheroma visualization.
Materials and Methods
Atheroma were induced in arteries of Yucatan miniswine (n = 5) by endothelial cell denudation followed by a 4-week high cholesterol diet. The contralateral arteries were left intact and served as controls. Anti-intercellular adhesion molecule-1 (ICAM-1) and generic gammaglobulin (IgG) conjugated ELIP were prepared. Arteries were imaged using IVUS before and after ELIP injection. Images were digitized, manually traced, segmented, and placed in tomographic sequence for 3D visualization. Atheroma brightness enhancement was compared and reported as mean gray scale values. Plaque volume was quantified both from IVUS and histologic images.
Anti-ICAM-1 ELIP highlighting of the atheroma in all arterial segments was different compared with baseline (P < 0.05). There was no difference in the mean gray scale values with IgG-ELIP. Arterial 3D IVUS images allowed visualization of the entire plaque distribution. The highlighted plaque/atheroma volume with anti-ICAM-1 ELIP was greater than baseline (P < 0.01).
This study demonstrates specific highlighting of early/inflammatory atheroma in vivo using anti-ICAM-1 ELIP. Three-dimensional IVUS reconstruction provides good visualization of plaque distribution in the arterial wall. This novel methodology may help to detect and diagnose pathophysiologic development of all stages of atheroma formation in vivo and quantitate plaque volume for serial and long-term atherosclerotic treatment studies.
atherosclerosis; intravascular ultrasound; liposomes; targeted; 3D reconstruction
Coronary anomalies are the cause of 12% of sudden deaths among athletes. Similarly anomalous coronary origin from the opposite sinus is often found at autopsy. The use of echocardiography to screen for these types of defects may provide a potentially life-saving diagnosis. The authors present a case that highlights the utility of echocardiography as part of a comprehensive screening program for athletes.
sudden cardiac death; anomalous origin of right coronary artery; anomalous coronary arteries; athlete screening; congenital heart disease
Targetable echogenic liposomes (ELIP) for ultrasound enhancement of atheroma have recently been developed; however, their retention of echogenicity at physiological temperature is less than desirable. The purpose of this study was to improve ELIP stability and increase clinical potential. The approach utilized the original procedures but involved manipulation of the lipid composition by reducing the level of unsaturation of the phospholipids components to minimize the rate of loss of echogenicity. Echogenicity was measured using a 20 MHz intravascular ultrasound (IVUS) catheter and quantified (as mean gray scale values) using computer-assisted videoden-sitometry. The optimal preparation for retention of echogenicity stability at physiologic temperature was egg phosphatidylcholine/dipalmitoylphosphatidylcholine/dipalmitoylphos-phatidylethanolamine/dipalmitoylphosphatidylglycerol/cholesterol (27:42:8:8:15, molar percent). This preparation retained 51 ± 3.5% of its echogenicity after 1 h at 37°C, more than 5× that retained by the previously descried preparation. In this composition nearly 2/3 of the phosphosphatidylcholine is fully saturated. Such an increase in saturation is anticipated to stiffen the lipid acyl chains. The air pockets that are responsible for reflection of ultrasound waves can be assumed to be stabilized by a lipid monolayer at the interface between the air and bulk water. The increased rigidity of that monolayer is presumed to be responsible for reducing the loss of air and extending the duration of echogenic activity. The stability of this improved formulation now appears to be more than adequate for clinical applications.
bilayer; ultrasound; stability; freeze drying/lyophilization; formulation; imaging methods; lipids; liposomes; phospholipids; physical stability
Echogenic liposomes (ELIP) have additional promise, beyond diagnostic agents, as vehicles for delivering oligonucleotides (ODN), especially if the release of the agent can be triggered and its uptake can be enhanced by ultrasound application at a specific site. The purpose of this study was to co-encapsulate air and NF-κB decoy ODN within ELIP allowing ultrasound to release encapsulated ODN from ELIP, and to accurately quantify release of encapsulated ODN from ELIP upon ultrasound application. FITC-labeled sense ODN (2 mM) was incorporated within ELIP using freeze/thaw method. Encapsulation efficiency of FITC-ODN was spectrofluorometrically analyzed by quenching fluorescence of unencapsulated FITC-ODN using a complementary strand tagged with Iowa Black FQ-ODN. Quenching of FITC-ODN (0.05 μM) with Iowa Black FQ-ODN (0.1 μM) was found to be efficient (92.4 ± 0.2 %), allowing accurate determination of encapsulated ODN. Encapsulation efficiency of ODN was 14.2 ± 2.5 % in DPPC/DOPC/DPPG/CH liposomes and 29.6 ± 1.5 % in DPPC/DOPE/DPPG/CH liposomes. Application of ultrasound (1 MHz continuous wave, 0.26 MPa peak-to-peak pressure amplitude, 60 seconds.) to the latter formulation triggered 41.6 ± 4.3 % release of ODN from ODN-containing ELIP. We have thus demonstrated that ODN can be encapsulated into ELIP and released efficiently upon ultrasound application. These findings suggest potential applications for gene therapy in atherosclerosis treatment.
Oligonucleotide; liposome; ultrasound; controlled release
The goal of this study was to determine whether targeted, Rhodamine-labeled echogenic liposomes (Rh-ELIP) containing nanobubbles could be delivered to the arterial wall, and whether 1 MHz continuous wave ultrasound would enhance this delivery profile. Aortae excised from apolipoprotein-E-deficient (n = 8) and wild-type (n = 8) mice were mounted in a pulsatile flow system through which Rh-ELIP were delivered in a stream of bovine serum albumin. Half the aortae from each group were treated with 1-MHz continuous wave ultrasound at 0.49 MPa peak-to-peak pressure, and half underwent sham exposure. Ultrasound parameters were chosen to promote stable cavitation and avoid inertial cavitation. A broadband hydrophone was used to monitor cavitation activity. After treatment, aortic sections were prepared for histology and analyzed by an individual blinded to treatment conditions. Delivery of Rh-ELIP to the vascular endothelium was observed, and subendothelial penetration of Rh-ELIP was present in five of five ultrasound-treated aortae and was absent in those not exposed to ultrasound. However, the degree of penetration in the ultrasound-exposed aortae was variable. There was no evidence of ultrasound-mediated tissue damage in any specimen. Ultrasound-enhanced delivery within the arterial wall was demonstrated in this novel model, which allows quantitative evaluation of therapeutic delivery.
ultrasound-assisted delivery; liposomes; ICAM-1 targeting; vascular endothelium; atheroma
To develop a new bioactive gas delivery method using echogenic liposomes (ELIP) as the gas carrier.
Nitric oxide (NO) is a bioactive gas with potent therapeutic effects. Bioavailability of NO by systemic delivery is low with potential systemic effects.
Liposomes containing phospholipids and cholesterol were prepared using a new freezing under pressure method. The encapsulation and release profile of NO from NO containing-ELIP (NO-ELIP) or a mixture of NO/Argon (NO/Ar-ELIP was studied. Uptake of NO from NO-ELIP by cultured vascular smooth muscle cells (VSMC) both in the absence and presence of hemoglobin was determined. The effect of NO-ELIP delivery to attenuate intimal hyperplasia in a balloon-injured artery was determined.
Coencapsulation of NO with argon (Ar) enabled the adjustment the amount of encapsulated NO. A total of 10 µl of gas can be encapsulated into 1 mg liposomes. The release profile of NO from NO-ELIP demonstrated an initial rapid release followed by a slower release over 8 hours. Sixty-eight percent of cells remained viable when incubated with 80 µg/ml of NO/Ar-ELIP for 4 hours. NO delivery to VSMC using NO/Ar-ELIP was 7-fold higher than unencapsulated NO. NO/Ar-ELIP remained effective NO delivery to VSMC even in the presence of hemoglobin. Local NO-ELIP administration to balloon-injured carotid arteries attenuated the development of intimal hyperplasia and reduced arterial wall thickening by 41±9%.
Liposomes can protect and deliver a bioactive gas to target tissues with the potential for both visualization of gas delivery and controlled therapeutic gas release.
Intimal hyperplasia; liposomes; nitric oxide; contrast agent
As the human lifespan becomes progressively extended, potential health-related effects of intense aerobic exercise after age 65 need evaluation. This study evaluates the cardiovascular (CV), pulmonary, and metabolic effects of competitive distance running on age-related deterioration in men between 69 (±3) and 77 (±2) years (mean ±SD). Twelve elderly competitive distance runners (ER) underwent oxygen consumption and echo/Doppler treadmill stress testing (Balke protocol) for up to 10 years. Twelve age-matched sedentary controls (SC) with no history of CV disease were similarly tested and the results compared for the initial three series of the study. CV data clearly separated the ER from SC. At entry, resting and maximal heart rate, systolic/diastolic blood pressure, peak oxygen consumption (VO2max), and E/A ratio of mitral inflow were better in the ER (P < 0.05 vs. SC). With aging, ER had a less deterioration of multiple health parameters. Exceptions were VO2max and left ventricular diastolic function (E/A, AFF, IVRT) that decreased (P < 0.05, Year 10 vs. Year 1). Health advantages of high-level aerobic exercise were demonstrated in the ER when compared to SC. Importantly, data collected in ER over 10 years confirm the benefit of intensive exercise for slowing several negative effects of aging. However, the normative drop of exercise capacity in the seventh and eighth decades reduces the potential athleticism plays in prevention of CV events.
aging; exercise; cardiac function; echocardiography
Vascular smooth muscle cells (VSMCs) are important targets in the treatment of atherosclerosis. However, the arterial media, where majority of VSMCs reside, has proven to be a difficult target for drug/gene delivery. We have demonstrated that ultrasound enhances drug/gene delivery to VSMCs in vitro using echogenic immunoliposomes (ELIP) as vector. This study aimed to evaluate whether ultrasound can similarly enhance delivery of an agent to VSMCs, particularly within the arterial media, in vivo, using ELIP. Anti-smooth muscle cell actin-conjugated calcein-loaded ELIP were injected into the peripheral arteries of Yucatan miniswine (n=8 arterial pairs). The right-sided porcine arteries were treated with 1-MHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.23 ± 0.05 MPa for two minutes. The contralateral arteries served as controls. Arteries were harvested after 30 minutes and imaged with fluorescence microscopy. Image data were converted to gray scale, and analyzed using computer-assisted videodensitometry. There was significant improvement in calcein uptake in all three arterial layers in the arteries exposed to ultrasound (p < 0.05 vs. no ultrasound), with a more marked increase in uptake in the arterial media (> 300%). This enhanced uptake was site specific and appeared limited to the ultrasound-treated arterial segment. We have demonstrated enhanced delivery of a small molecule to VSMCs in all arterial wall layers particularly the arterial media, using ultrasound and targeted ELIP. The combined effect of ultrasound exposure and ELIP as a contrast agent and a drug/gene-bearing vector has the potential for site-specific therapy directed at VSMC function.
Ultrasound Contrast Agents; Targeted Delivery
Constriction and relaxation of peripheral conduit arteries in response to exercise and recovery are amenable to noninvasive imaging. Diameter changes in the brachial artery during stationary bicycle pedaling are paradoxical to those in the lower extremities. When exercise is confined to the legs, arteries in the upper extremities constrict while leg arteries dilate. The magnitude of vasoconstriction in the upper extremities reflects the integrity of exercise mediated vascular responses. In the current study, young and old men with various levels of physical activity and cardiovascular histories exercised on a stationary bicycle while brachial artery diameter and flow velocity were continuously obtained. Results suggest that normal aging blunts arterial reactivity in seniors even if they exercise regularly. However, arterial dysfunction is greater when associated with sedentary lifestyle. This methodology of imaging brachial artery diameter changes during bicycle pedaling appears to be an effective tool for assessing the physiologic integrity of the vascular bed.
exercise; vascular imaging; elderly
Intrinsically echogenic liposomes (ELIP) can be adapted to encapsulate nitric oxide to facilitate ultrasound-enhanced delivery of therapeutic agents to atherosclerotic plaques. However, the NO loading of targeted ELIP caused a 93% decrease of antibody (Ab) immunoreactivity. The following hypothesis was tested: biotin/avidin-mediated coupling of NO-ELIP and Ab-conjugated ELIP will enable co-delivery of bioactive gases and ELIP that can encapsulate other agents without loss of targeting efficiency. Complex formation was initiated by addition of excess streptavidin to equal proportions of biotinylated Ab-ELIP and NO-ELIP. Fluorescence deconvolution microscopy, Coulter Multisizer 3 analysis and flow cytometry demonstrated that the ELIP coupling procedure formed mixed aggregates of ≥10 liposomes within 1 minute. Intravascular ultrasound imaging and ELISA showed that echogenicity and targeting efficiency were completely and 69–99% retained, respectively. When complexed to NO-ELIP, ELIP bifunctionally targeted to both CD34 and ICAM-1 (BF-ELIP) increased human mononuclear cell migration through human coronary artery endothelial cell monolayers in transwell plates 4-fold relative to a nonspecific IgG-ELIP control and 2-fold relative to BF-ELIP alone. It was concluded that this novel multi-functional conjugation methodology provides a platform technology for site-specific co-delivery of bioactive gases and other agents.
Liposomes; Bioactive Gases; Atherosclerosis; Stem Cells; Ultrasound
Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.
Echogenic liposomes (ELIP) were developed as ultrasound-triggered targeted drug or gene delivery vehicles (Lanza et al., 1997; Huang et al., 2001). Recombinant tissue-type Plasminogen Activator (rt-PA), a thrombolytic, has been loaded into ELIP (Tiukinhoy-Laing et al., 2007). These vesicles have the potential to be used for ultrasound-enhanced thrombolysis in the treatment of acute ischemic stroke, myocardial infarction, deep vein thrombosis, or pulmonary embolus. A clinical diagnostic ultrasound scanner (Philips HDI 5000) equipped with a linear array transducer (L12-5) was employed for in vitro studies using rt-PA-loaded ELIP (T-ELIP). The goal of this study was to quantify ultrasound-triggered drug release from rt-PA-loaded echogenic liposomes. T-ELIP samples were exposed to 6.9-MHz B-mode pulses at a low pressure amplitude (600 kPa) to track the echogenicity over time under four experimental conditions: 1) flow alone to monitor gas diffusion from the T-ELIP, 2) pulsed 6.0-MHz color Doppler exposure above the acoustically driven threshold (0.8 MPa) to force gas out of the liposome gently, 3) pulsed 6.0-MHz color Doppler above the rapid fragmentation threshold (2.6 MPa), or 4) Triton X-100 to rupture the T-ELIP chemically as a positive control. Release of rt-PA for each ultrasound exposure protocol was assayed spectrophotometrically. T-ELIP were echogenic in the flow model (5 ml/min) for thirty minutes. The thrombolytic drug remained associated with the liposome when exposed to low-amplitude B-mode pulses over 60 min and was released when exposed to color Doppler pulses or Triton X-100. The rt-PA released from the liposomes had similar enzymatic activity as the free drug. These T-ELIP are robust and echogenic during continuous fundamental 6.9-MHz B-mode imaging at a low exposure output level (600 kPa). Furthermore, a therapeutic concentration of rt-PA can be released by fragmenting the T-ELIP with pulsed 6.0-MHz color Doppler ultrasound above the rapid fragmentation threshold (1.59 MPa).
ultrasound-enhanced thrombolysis; drug delivery; therapeutic ultrasound; echogenic liposomes; ultrasound contrast agents; destruction thresholds