Patrick Kee

Impact of Short-Term Administration of High-Density Lipoproteins and Atorvastatin on Atherosclerosis in Rabbits

Objective—This study investigates effects of short-term administration of high-density lipoproteins (HDL) and a statin on atherosclerosis in cholesterol-fed rabbits. Effects of HDL apolipoprotein and phospholipid composition have also been investigated. Methods and Results—Aortic atherosclerosis was established over 17 weeks in 46 rabbits by balloon denudation and cholesterol feeding. During the past 5 days of the cholesterol-feeding period, animals received: (1) no treatment; (2) oral atorvastatin 5 mg/kg on each of the 5 days; or (3) infusions of HDL (8 mg/kg apolipoprotein A-I) on days 1 and 3 of the treatment phase. After euthanization, lesion size and composition were assessed by histological and immunohistochemical analysis. HDL (but not atorvastatin) reduced lesion size by 36% (P<0.05). The ratio of smooth muscle cells to macrophages in the lesions increased 2.6-fold in animals infused with HDL (P<0.05) and 4-fold in those receiving atorvastatin (P<0.01). HDL and atorvastatin reduced matrix metalloproteinase (MMP)-9 expression by 42% (P<0.05) and 45% (P<0.03), respectively. HDL increased thrombomodulin expression 2-fold (P<0.03). The beneficial effects on lesion area and plaque cellular composition were influenced by HDL phospholipid and apolipoprotein composition. Conclusion—Infusing small amounts of HDL rapidly reduces lesion size and is comparable to atorvastatin in promoting a stable plaque phenotype.

Nitric Oxide Loaded Echogenic Liposomes for Nitric Oxide Delivery and Inhibition of Intimal Hyperplasia

OBJECTIVES We sought to develop a new bioactive gas-delivery method by the use of echogenic liposomes (ELIP) as the gas carrier. BACKGROUND Nitric oxide (NO) is a bioactive gas with potent therapeutic effects. The bioavailability of NO by systemic delivery is low with potential systemic effects. METHODS Liposomes containing phospholipids and cholesterol were prepared by the use of a new method, freezing under pressure. The encapsulation and release profile of NO from NO-containing ELIP (NO-ELIP) or a mixture of NO/argon (NO/Ar-ELIP) was studied. The uptake of NO from NO-ELIP by cultured vascular smooth muscle cells (VSMCs) 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. RESULTS Coencapsulation of NO with Ar enabled us to adjust the amount of encapsulated NO. A total of 10 microl of gas can be encapsulated into 1 mg of liposomes. The release profile of NO from NO-ELIP demonstrated an initial rapid release followed by a slower release during the course of 8 h. Sixty-eight percent of cells remained viable when incubated with 80 microg/ml of NO/Ar-ELIP for 4 h. The delivery agent of NO to VSMCs by the use of NO/Ar-ELIP was 7-fold greater than unencapsulated NO. We discovered that NO/Ar-ELIP remained an effective delivery agent of NO to VSMCs 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%. CONCLUSIONS 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.

Ultrasound-Mediated Release of Hydrophilic and Lipophilic Agents From Echogenic Liposomes

Objective. To achieve ultrasound‐controlled drug delivery using echogenic liposomes (ELIPs), we assessed ultrasound‐triggered release of hydrophilic and lipophilic agents in vitro using color Doppler ultrasound delivered with a clinical 6‐MHz compact linear array transducer. Methods. Calcein, a hydrophilic agent, and papaverine, a lipophilic agent, were each separately loaded into ELIPs. Calcein‐loaded ELIP (C‐ELIP) and papaverine‐loaded ELIP (P‐ELIP) solutions were circulated in a flow model and treated with 6‐MHz color Doppler ultrasound or Triton X‐100. Treatment with Triton X‐100 was used to release the encapsulated calcein or papaverine content completely. The free calcein concentration in the solution was measured directly by spectrofluorimetry. The free papaverine in the solution was separated from liposome‐bound papaverine by spin column filtration, and the resulting papaverine concentration was measured directly by absorbance spectrophotometry. Dynamic changes in echogenicity were assessed with low‐output B‐mode ultrasound (mechanical index, 0.04) as mean digital intensity. Results. Color Doppler ultrasound caused calcein release from C‐ELIPs compared with flow alone (P < .05) but did not induce papaverine release from P‐ELIPs compared with flow alone (P > .05). Triton X‐100 completely released liposome‐associated calcein and papaverine. Initial echogenicity was higher for C‐ELIPs than P‐ELIPs. Color Doppler ultrasound and Triton X‐100 treatments reduced echogenicity for both C‐ELIPs and P‐ELIPs (P < .05). Conclusions. The differential efficiency of ultrasound‐mediated pharmaceutical release from ELIPs for water‐ and lipid‐soluble compounds suggests that water‐soluble drugs are better candidates for the design and development of ELIP‐based ultrasound‐controlled drug delivery systems.

In Vivo Therapeutic Gas Delivery for Neuroprotection With Echogenic Liposomes

Background— Ischemia-related neurological 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 intra-arterially after right middle cerebral artery occlusion. One-megahertz 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 neurological injury and disability were evaluated. Xe-ELIP delivery to oxygen/glucose-deprived neuronal cells improved cell viability in vitro and resulted in a 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 after intra-arterial administration. Behavioral tests on limb placement and grid and beam walking correlated with infarct reduction. Conclusions— 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.

Metabolism of ApoA-I as Lipid-Free Protein or as Component of Discoidal and Spherical Reconstituted HDLs: Studies in Wild-Type and Hepatic Lipase Transgenic Rabbits

Objective—Apolipoprotein (apo)A-I exists in 3 forms in plasma: as lipid-free apoA-I, as a component of pre–&bgr;-migrating discoidal high density lipoproteins (HDLs), and as a component of &agr;-migrating spherical HDLs. This study investigates (1) the in vivo metabolism of apoA-I in each of these forms and (2) the effects of hepatic lipase (HL) on apoA-I metabolism. Methods and Results—Wild-type and HL transgenic rabbits were studied. When lipid-free 125I-apoA-I and 125I-apoA-I in pre–&bgr;-migrating discoidal reconstituted HDLs (rHDLs) were injected into wild-type rabbits, the label rapidly appeared in &agr;-migrating particles and decayed with the same fractional catabolic rate (FCR) as when they were injected as a component of spherical rHDLs. Spherical rHDLs did not change in size when they were injected into wild-type rabbits but were reduced in size in HL transgenic rabbits. The FCR of apoA-I in HL transgenic rabbits was double that in wild-type rabbits. Conclusions—In vivo, (1) lipid-free apoA-I rapidly incorporates into preexisting &agr;-migrating particles, (2) pre–&bgr;-migrating discoidal HDLs are rapidly converted into &agr;-migrating HDLs, (3) the FCR of apoA-I is independent of the form in which it is introduced into plasma, and (4) HL reduces the size of &agr;-migrating HDLs and increases the rate of catabolism of apoA-I.

Ultrasound-Enhanced Thrombolytic Effect of Tissue Plasminogen Activator–Loaded Echogenic Liposomes in an In Vivo Rabbit Aorta Thrombus Model—Brief Report

Objective—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 &mgr;g 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). Conclusion—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.

Effect of Inhibiting Cholesteryl Ester Transfer Protein on the Kinetics of High-Density Lipoprotein Cholesteryl Ester Transport in Plasma In Vivo Studies in Rabbits

Objective—Inhibitors of cholesteryl ester transfer protein (CETP) have been developed as potential anti-atherogenic agents. Theoretically, however, they may be pro-atherogenic by blocking one of the pathways for removing high-density lipoprotein (HDL) cholesteryl esters (CE) from plasma in the final step of reverse cholesterol transport. Here we describe how CETP inhibition in rabbits impacts on the kinetics of HDL CE transport in plasma. Methods and Results—Administration of a CETP inhibitor reduced CETP activity by 80% to 90% and doubled the HDL cholesteryl ester concentration. Multi-compartmental analysis was used to determine HDL CE kinetics in CETP-inhibited and control rabbits after injection of tracer amounts of both native and reconstituted HDL labeled with 3H in the CE moiety. In control rabbits, HDL CE was removed from plasma by both a direct pathway and an indirect pathway after transfer of HDL CE to the very-low-density lipoprotein/low-density lipoprotein fraction. In CETP-inhibited rabbits there was an almost complete block in removal via the indirect pathway. This did not compromise the overall removal of HDL CE from plasma, which was not different in control and inhibited animals. Conclusion—Inhibiting CETP in rabbits does not compromise the removal of HDL CE from plasma.

CT imaging of myocardial scars with collagen-targeting gold nanoparticles.

UNLABELLED In the setting of myocardial ischemia, recovery of myocardial function by revascularization procedures depends on the extent of coronary disease and myocardial scar burden. Currently, computed tomographic (CT) imaging offers superior evaluation of coronary lesions but lacks the capability to measure the transmural extent of myocardial scar. Our work focuses on determining if collagen-targeting gold nanoparticles (AuNPs) can effectively target myocardial scar and provide adequate contrast for CT imaging. AuNPs were coated with a collagen-homing peptide, collagen adhesin (CNA35). Myocardial scar was created in mice by occlusion/reperfusion of the left anterior descending coronary artery. Thirty days later, un-gated CT imaging was performed. Over 6h, CNA35-AuNPs provided uniform and prolonged opacification of the vascular structures (100-130 HU). In mice with larger scar burden, focal contrast enhancement was detected in the myocardium, which was not apparent within that of control mice. Histological staining confirmed myocardial scar formation and accumulation of AuNPs. FROM THE CLINICAL EDITOR This team of investigators presents a collagen-targeting gold nanoparticle-based approach that enables the imaging of myocardial scars via CT scans in a rodent model. This information would enable clinicians to judge the recovery potential of myocardium more accurately than the current CT-scan based approaches.

In vivo volumetric intravascular ultrasound visualization of early/inflammatory arterial atheroma using targeted echogenic immunoliposomes.

Objectives: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. Results: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). Conclusion: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.

Evaluation of Mitral Valve Dynamics

Mitral valve (mv) repair is the preferred treatment for patients with mv insufficiency. The unsolved problem in MV repair surgery is predicting the optimal repair for each patient. This is in part due to lack of physiological imaging modalities to provide this information prior to or at the time of valve repair. Moreover, the majority of cases have complex pathophysiological involvement including annular enlargement, chordal lengthening, chordal rupture, calcification, and ultimately lack of proper leaflet coaptation. Current clinical 3-dimensional (3D) transesophageal echocardiography (TEE) can demonstrate volumetric morphology of the MV apparatus. However, biomechanical information is not available from 3D echocardiography. If imaging techniques can be combined with appropriate computational MV evaluation methods, then improved diagnosis and therapeutic approaches to MV repair can be developed. In the present study, we describe a novel comprehensive evaluation protocol to improve diagnosis and treatment of MV pathology by combining 3D TEE and computational simulation techniques (Fig. 1). Virtual MV models were created by utilizing 3D TEE data of patients with normal and pathological MVs followed by computational simulations of MV function. Computational simulations clearly demonstrated deformation and stress distribution of the MV structure across the cardiac cycle at a microsecond scale and corresponded well to 3D TEE data (Figs. 2 and ​and3,3, Online Videos 1, 2, 3, and 4). Here we present 4 case studies (1 normal and 3 different types of pathological MVs). Figure 1 Flow Chart for Virtual MV Modeling and Computational Simulation of MV Function Figure 2 Images of a Normal MV Figure 3 Images of a Pathological MV With Posterior Chordal Rupture Case 1 (Fig. 4): a normal MV demonstrated complete coaptation with no regurgitation when closed. Figure 4 Case 1: A Normal MV With Complete Coaptation; A to D (Peak Systole), E to H (End Diastole) Case 2 (Fig. 5): this MV showed mild regurgitation with relatively small annular dilation. Computational simulation indicated increased leaflet stress values and reduced contact pressure between the leaflets. Figure 5 Case 2: A Degenerative MV With Mild Regurgitation and Small Annular Dilation; A to D (Peak Systole), E to H (End Diastole) Case 3 (Fig. 6): a degenerative MV with large annular dilation demonstrated severe regurgitation by 3D Doppler TEE and the lesion corresponded to regions with no leaflet contact in the computational simulation. Figure 6 Case 3: A Degenerative MV With Severe Regurgitation and Large Annular Dilation; A to D (Peak Systole), E to H (End Diastole) Case 4 (Fig. 7): chordal rupture of this MV caused posterior leaflet prolapse inducing the regurgitant jet. Computational simulation demonstrated an extremely asymmetric and large stress distribution over the leaflets and lack of leaflet coaptation in the regurgitant region. Figure 7 Case 4: A Degenerative MV With Severe Regurgitation due to Ruptured Chordae; A to D (Peak Systole), E to H (End Diastole) Comparative studies of the normal MV (Case 1) and the pathological MV with ruptured chordae (Case 4) clearly demonstrated differences in annular reaction forces and chordal stresses (Fig. 8), and in the degree of leaflet coaptation (Fig. 9, Online Videos 5 and 6). Figure 8 Assessment of Annular Reaction Force and Chordal Stress Distribution; Normal MV (Case 1) Versus MV With Ruptured Chordae (Case 4) Figure 9 Contact Pressure Distribution Between the Leaflets; Normal MV (Case 1) Versus MV With Ruptured Chordae (Case 4) Although MV morphology obtained with 3D TEE image data may demonstrate relatively normal function with no regurgitation, the leaflets may be under extremely high stresses which can result in annular dilation and MV deterioration. Biomechanical information from computational simulation further provides information to help better understand MV pathophysiology. This novel computational strategy has the potential to predict pathophysiological alterations in MV structure, help cardiologists to quantitatively evaluate the extent and severity of MV pathology, and help surgeons to better understand MV dynamics before and following repair to determine more suitable patient-specific repair techniques.