Rw Renate Boekhoven

Anti-tumor activity of liposomal glucocorticoids: The relevance of liposome-mediated drug delivery, intratumoral localization and systemic activity.

Tumor-associated inflammation has been recognized as an important tumor growth propagator and, therefore, represents an attractive target for anti-cancer therapy. In the current study, inspired by recent findings on the anti-tumor activity of liposomal glucocorticoids, we introduce paramagnetic and fluorescent liposomes, encapsulating prednisolone phosphate (PLP), to evaluate the local delivery of liposomal glucocorticoids to the tumor and its importance for the therapeutic response. The new multifunctional liposomes (Gd-PLP-L) (120nm diameter, 5.8mg PLP/60μmol lipid, bioexponential blood-clearance kinetics (T(1/2α)=2.4±0.5h, T(1/2β)=42.0±12.4h), drug leakage of 15%/72h (in vitro)), containing 25mol% Gd-DTPA-lipid and 0.1mol% of rhodamine-lipid, were tested in B16F10 melanoma subcutaneously inoculated in C57BL/6 mice, and compared to the original PLP formulation (PLP-L). A single dose of Gd-PLP-L (20mgPLP/kg/week, i.v.) was found to significantly inhibit tumor growth compared to non-treated mice (P<0.05), similarly to PLP-L. The accumulation efficacy of the liposomal agent in the tumor was assessed with MRI, using the increase in the longitudinal relaxation rate (ΔR(1)) as a marker. Interestingly, large inter-tumor differences in ΔR(1) (0.009-0.063s(-1), 24h post-administration), corresponding to highly variable intratumoral Gd-PLP-L levels, did not correlate to the effectiveness of tumor growth inhibition. Uptake of liposomes by tumor-associated macrophages (TAM), determined by ex-vivo fluorescence microscopy, was limited to only 5% of the TAM population. Furthermore, the therapy did not lead to TAM depletion. Importantly, a 90% drop in white blood cell count both after Gd-PLP-L and PLP-L administration was observed. This depletion may reduce tumor infiltration of monocytes, which stimulate angiogenesis, and, thus, possibly co-contributes to the anti-tumor effects. In conclusion, MRI provides a powerful instrument to monitor the delivery of liposomal therapeutics to tumors and guided us to reveal that the activity of liposomal glucocorticoids is not limited to the tumor site only.

ECHO-COMPUTED TOMOGRAPHY STRAIN IMAGING OF HEALTHY AND DISEASED CAROTID SPECIMENS

To improve our understanding of the mechanical behavior of human atherosclerotic plaque tissue, fully 3-D geometrical, morphological and dynamical information is essential. For this purpose, four-dimensional (3-D+t) strain imaging using an ultrasound tomography approach (echo-computed tomography) was performed in carotid arteries in vitro. The method was applied to a carotid phantom (CPh), a porcine carotid artery (PC) and human carotid atherosclerotic plaque samples (HC, n = 5). Each sample was subjected to an intraluminal pressure, after which 2-D longitudinal ultrasound images were obtained for 36 angles along the circumferential direction. Local deformations were estimated using a 2-D strain algorithm, and 3-D radial strain data were reconstructed. At systole, median luminal strains of 15% (CPh) and 18% (PC) were found, which is in agreement with the stiffness of the material and applied pressure pulse. The elastographic signal-to-noise ratio was consistent in all directions and ranged from 16 to 36 dB. Furthermore, realistic but more complex strain patterns were found for the HC, with 99th percentile systolic strain values ranging from 0.1% to 18%.

Towards mechanical characterization of intact endarterectomy samples of carotid arteries during inflation using Echo-CT

In this study, an experimental framework is described that allows pressurization of intact, human atherosclerotic carotid samples (inflation testing), in combination with ultrasound imaging. Eight fresh human carotid endarterectomy samples were successfully pressurized and tested. About 36 2-D (+t) ultrasound datasets were acquired by rotating the vessel in 10° steps (Echo-CT), from which both 3-D geometry and 3-D strain data were obtained. Both geometry and morphology were assessed with micro-CT imaging, identifying calcified and lipid rich regions. US-based and CT-based geometries were matched for comparison and were found to show good agreement, with an average similarity index of 0.71. Realistic pressure-volume relations were found for 6 out of 9 samples. 3-D strain datasets were reconstructed, revealing realistic strain patterns and magnitudes, although the data did suffer from a relatively high variability. The percentage of fat and calcifications (micro-CT) were compared with the median, 75th and 99th percentile strain values (Echo-CT). A moderate trend was observed for 75th and 99th percentile strains, higher strains were found for more lipid rich plaques, where lower strains were found for highly calcified plaques. However, an inverse numerical modeling technique is necessary for proper mechanical characterization the of plaque components, using the geometry, morphology and wall deformation as input.

A novel experimental approach for three-dimensional geometry assessment of calcified human stenotic arteries in vitro

To improve diagnosis and understanding of the risk of rupture of atherosclerotic plaque, new strategies to realistically determine mechanical properties of atherosclerotic plaque need to be developed. In this study, an in vitro experimental method is proposed for accurate 3-D assessment of (diseased) vessel geometry using ultrasound. The method was applied to a vascular phantom, a healthy porcine carotid artery and human carotid endarterectomy specimens (n = 6). Vessel segments were pressure fixed and rotated in 10 ° steps. Longitudinal cross sections were imaged over 360 °. Findings were validated using micro-computed tomography (μCT). Results show good agreement between ultrasound and μCT-based geometries of the different segment types (ISI phantom = 0.94, ISI healthy = 0.79, ISI diseased = 0.75-0.80). The method does not suffer from acoustic shadowing effects present when imaging stenotic segments and allows future dynamic measurements to determine mechanical properties of atherosclerotic plaque in an in vitro setting.

Design of a fatty plaque phantom for validation of strain imaging

Prior to clinical application of novel techniques such as vascular elastography and photo acoustic imaging, validation studies are of great importance. Gelatin, agar and polyvinyl-alcohol (PVA) phantoms are commonly used. However, more realistic phantoms are needed with complex geometry and different constituents with known properties. For this purpose, a fatty plaque phantom (fPP) was designed. A 15 weight percent (wt%) PVA solution was made, and 1 wt% of silica scatters were added (3 8 nm). The solution was poured in to a custom made mold, acquiring the fPP in four freeze/thaw cycles. B-mode data were acquired using a MyLab70 (Esaote, NL), while being pressurized from 0-80 mmHg. The phantom was imaged using a 3D echo-CT strain imaging method [1], [2], to determine 3D geometry, distension and radial strains. In accordance with the distension data, higher radial strains were found at the lipid inclusion location. In future work, strain data will be compared to numerical models that match the phantoms and experimental conditions to validate the strains measured. Additional stiffening of the PVA using a coagulation bath will be explored, by doing so, increasing the systolic pressure allowed.

In vitro elastography of porcine carotid arteries, a validation study

Reliable non-invasive assessment of mechanical properties of the arterial wall, either healthy or diseased, is of great importance for patient specific diagnosis in the field of vascular disease. Vascular elastography is a promising tool to assess these mechanical properties. Therefore, in this study, the most controllable and realistic approach of mechanical testing of arteries in vitro, inflation testing, is compared to the gold standard of mechanical testing, bi-axial testing.

Non-invasive strain imaging in normotensive and hypertensive patients

Literature shows that arterial stiffness is related to age and degree of atherosclerosis. It has been hypothesized that there is no difference in arterial stiffness between normotensive (NT) and hypertensive (HT) patients of same sex and age. This was demonstrated in a previous study by analysing pressure diameter curve data and estimating the incremental Youngs modulus [1]. In this study, 2D strain imaging was used to investigate possible differences in carotid stiffness by quantifying the response to pressure, i.e., strain, in a group of NT and HT subjects, matched for both age and sex. Radio-frequency data of the common carotid artery were acquired in 18 NT and 18 HT patients, with a mean age of 52 ± 11, using the Mylab 70 (Esaote, NL). Displacements and strains were estimated using a 2D strain imaging algorithm [2]. Strain analysis was performed and on the far wall data at end-systole. One-way ANOVA indicates significant lower strains in the HT groups throughout the vessel wall (NT = 6.3%±5.6%; HT = 4.8%±3.9%). The lower strain and distensibility can be explained by the higher mean arterial pressure and non-linear behaviour of the vascular tissue. These results confirm the hypothesis that the stiffness in HT patients is not necessarily higher compared to NT patients.

Geometrical and Morphological Assessment of Human Endarterectomy Specimens In Vitro

Treatment of rupture-prone carotid atherosclerotic plaques, by means of endarterectomy, is only beneficial for patients with unstable plaques, which comprise only 16% of the patient population [1]. It is therefore of great interest to assess morphology, geometry and mechanical deformation of the plaque and its components, to prevent unnecessary treatment. However, due to the complex geometry of stenotic arteries, 3D information at both high temporal and spatial resolution is required. Besides, assessment of plaque morphology in vivo can still not be routinely performed. Therefore, one has to rely on in vitro methods to obtain morphology and mechanical properties, and thus rupture risk.Copyright

Flow induced vasodilation in porcine carotid arteries

Endothelial cells play an important role in the autoregulation of the vascular diameter for maintaining physiological flow and shear stress. An increase in blood flow causes an increase in shear stress, which is sensed by the endothelial cells, resulting in the release of vasoactive substances. An impaired endothelial mediated vasomotive response seems reflective for several cardiovascular pathologies, such as failure of atherosclerosis and arterio-venous fistulas (AVF).Copyright

Novel Strategy of the Determination of Mechanical Properties of Human Carotid Atherosclerotic Plaques

Carotid endarterectomy is the procedure of choice in patients with a recent symptomatic stenosis of 70–99%. Currently, the selection of candidates eligible for carotid endarterectomy is based on stenosis size only. However, the treatment is only beneficial for patients with unstable plaques, which comprises only 16% of the patient population [1]. Hence, identifying plaque stability at an early stage would permit timely intervention, while substantially reducing overtreatment of stable plaques. The objective of this study is to distinguish between stable and unstable carotid atherosclerotic plaques by determining the plaque geometry, the plaque composition and the mechanical properties of plaque components in three dimensions (3D). Mechanical properties from healthy vessels were assessed earlier by van den Broek et al. [2] using ultrasound (US) imaging. They obtained a dynamic dataset in 2D + t. When blood pressure and vessel wall movement are known, mechanical properties can be extracted from these data using a constitutive model. However, atherosclerotic plaques are mostly asymmetric, and present calcifications will cause unfavorable acoustic shadowing when using US. In this study, the focus is on the assessment of plaque geometry, from in vitro echo-CT data, overcoming the aforementioned problems. In an experimental set-up (Fig. 1) both healthy and endarterectomy specimens were mounted, and exposed to physiological intraluminal pressures. Echo-CT was used to image the arterial segments in 3D+t. Automated geometry assessment of the arterial segments will be demonstrated and validated using microCT (μCT).Copyright