Koning, G.A.

In vivo comparison of arterial lumen dimensions assessed by co-registered three-dimensional (3D) quantitative coronary angiography, intravascular ultrasound and optical coherence tomography (Article)

2012-01-19T00:00:00Z

This study sought to compare lumen dimensions as assessed by 3D quantitative coronary angiography (QCA) and by intravascular ultrasound (IVUS) or optical coherence tomography (OCT), and to assess the association of the discrepancy with vessel curvature. Coronary lumen dimensions often show discrepancies when assessed by X-ray angiography and by IVUS or OCT. One source of error concerns a possible mismatch in the selection of corresponding regions for the comparison. Therefore, we developed a novel, real-time co-registration approach to guarantee the point-to-point correspondence between the X-ray, IVUS and OCT images. A total of 74 patients with indication for cardiac catheterization were retrospectively included. Lumen morphometry was performed by 3D QCA and IVUS or OCT. For quantitative analysis, a novel, dedicated approach for co-registration and lumen detection was employed allowing for assessment of lumen size at multiple positions along the vessel. Vessel curvature was automatically calculated from the 3D arterial vessel centerline. Comparison of 3D QCA and IVUS was performed in 519 distinct positions in 40 vessels. Correlations were r = 0.761, r = 0.790, and r = 0.799 for short diameter (SD), long diameter (LD), and area, respectively. Lumen sizes were larger by IVUS (P < 0.001): SD, 2.51 ± 0.58 mm versus 2.34 ± 0.56 mm; LD, 3.02 ± 0.62 mm versus 2.63 ± 0.58 mm; Area, 6.29 ± 2.77 mm2versus 5.08 ± 2.34 mm2. Comparison of 3D QCA and OCT was performed in 541 distinct positions in 40 vessels. Correlations were r = 0.880, r = 0.881, and r = 0.897 for SD, LD, and area, respectively. Lumen sizes were larger by OCT (P < 0.001): SD, 2.70 ± 0.65 mm versus 2.57 ± 0.61 mm; LD, 3.11 ± 0.72 mm versus 2.80 ± 0.62 mm; Area 7.01 ± 3.28 mm2versus 5.93 ± 2.66 mm2. The vessel-based discrepancy between 3D QCA and IVUS or OCT long diameters increased with increasing vessel curvature. In conclusion, our comparison of co-registered 3D QCA and invasive imaging data suggests a bias towards larger lumen dimensions by IVUS and by OCT, which was more pronounced in larger and tortuous vessels.

Angiogenesis: A prognostic determinant in pancreatic cancer? (Article)

2011-11-01T00:00:00Z

Angiogenesis has been associated with disease progression in many solid tumours, however the statement that tumours need angiogenesis to grow, invade and metastasise seems no longer applicable to all tumours or to all tumour subtypes. Prognostic studies in pancreatic cancer are conflicting. In fact, pancreatic cancer has been suggested an example of a tumour in which angiogenesis is less essential for tumour progression. The aim of the present study was therefore to measure angiogenesis in two anatomically closely related however prognostically different types of pancreatic cancer, pancreatic head and periampullary cancer, and investigate its relation with outcome. Vessels were stained by CD31 on original paraffin embedded tissue from 206 patients with microscopic radical resection (R0) of pancreatic head (n = 98) or periampullary cancer (n = 108). Angiogenesis was quantified by microvessel density (MVD) and measured by computerised image analysis of three randomly selected fields and investigated for associations with recurrence free survival (RFS), cancer specific survival (CSS), overall survival (OS) and conventional prognostic factors. MVD was heterogeneous both between and within tumours. A higher MVD was observed in periampullary cancers compared with pancreatic head cancers (p <.01). Furthermore, MVD was associated with lymph node involvement in pancreatic head (p =.014), but not in periampullary cancer (p =.55). Interestingly, MVD was not associated with RFS, CSS or with OS. In conclusion, angiogenesis is higher in periampullary cancer and although associated with nodal involvement in pancreatic head cancer, pancreatic cancer prognosis seems indeed angiogenesis independent.

Post-natal myogenic and adipogenic developmental:Defects and metabolic impairment upon loss of a-type lamins (Article)

2011-07-18T00:00:00Z

A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early postnatal development is poorly understood. Here we developed a novel LMNA null mouse (LMNAGT-/-) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early postnatal development critically contributes to the disease phenotypes in adult laminopathies.

Spin-echo small angle neutron scattering analysis of liposomes and bacteria (Article)

2010-12-01T00:00:00Z

Two types of liposomes, commonly used in drug delivery studies, and E. coli bacteria, all prepared in H2O, were resuspended in D2O and measured with Small Angle Spin-Echo Neutron Scattering (SESANS). Modeling was performed using correlation functions for solid spheres and hollow spheres. The signal strength and curve shape were more indicative of hollow particles, indicating that the H2O-D2O exchange occurred too fast to be observed with the available time resolution. Fitting the particle diameter and membrane thickness of the hollow sphere model to the data, gave results which were in good agreement with Dynamic Light Scattering (DLS) data and literature, showing as a proof-of-principle that SESANS is able to investigate such systems. SESANS may become a good alternative to conventional tritium studies or a tool with which to study intracellular vesicle transport phenomena, with possible in vivo applications. Calculations show that a substantial change in numbers of a mixed system of small and large biological particles should be observable. A possible application is the destruction by external means of great numbers of liposomes in the presence of tumor cells for triggered drug release in cancer treatment. Since SESANS is both non-invasive and non-destructive and can handle relatively thick samples, it could be a useful addition to more conventional techniques.

Differential expression and prognostic value of HMGA1 in pancreatic head and periampullary cancer (Article)

2010-12-01T00:00:00Z

The high mortality rate and minimal progress made in the treatment of pancreatic cancer over the last few decades, warrant an alternative approach. Treatment protocols should be individualised to the patient guided by prognostic markers. A particularly interesting target would be the architectural transcription factor high mobility group A1 (HMGA1), that is low or undetectable in normal tissue, induced during neoplastic transformation and consequently often exceptionally high in cancer. The aim of the current study was therefore to determine the differential expression of HMGA1 in pancreatic head and periampullary cancer and investigate its relation with outcome. HMGA1 expression was determined by immunohistochemistry on original paraffin embedded tissue from 99 pancreatic head- and 112 periampullary cancers (with R0). Expression was investigated for associations with recurrence free (RFS), cancer specific (CSS) and overall survival (OS) and conventional prognostic factors. HMGA1 was expressed in 47% and 26% of pancreatic head- and periampullary cancer, respectively and associated with poor RFS, CSS and OS in periampullary cancer. CSS 5 years following surgery was 25% and 44% for patients with tumours which were positive or negative for HMGA1 protein, respectively. HMGA1 expression was not associated with survival in pancreatic head cancer. In conclusion HMGA1 was identified as an independent prognostic marker predicting poor outcome in periampullary cancer. Although expressed to a higher extent as compared to periampullary cancer, HMGA1 was not associated with survival in pancreatic head cancer.

Thermosensitive liposomes for the delivery of cancer therapeutics (Article)

2010-11-01T00:00:00Z

Multimodality imaging reveals a gradual increase in matrix metalloproteinase activity at aneurysmal lesions in live fibulin-4 mice (Article)

2010-09-01T00:00:00Z

Background-We imaged the protease activity of matrix metalloproteinases (MMPs) upregulated during aneurysm formation, using protease-activatable near-infrared fluorescence probes. We tested whether these protease-activatable sensors can directly report the in vivo activity of the key biomarkers in aneurysm, using our genetically modified fibulin-4 mouse models for aneurysm formation. Mice homozygous for the fibulin-4 reduced-expression allele (fibulin-4R/R) show dilatation of the ascending aorta and a tortuous, stiffened aorta resulting from disorganized elastic fiber networks. Strikingly, even a moderate reduction in expression of fibulin-4 in the heterozygous fibulin-4+/Rmice occasionally results in modest aneurysm formation. Methods and Results-Aorta transcriptome and protein expression analysis of fibulin-4+/Rand fibulin-4R/Ranimals identified excessive transforming growth factor-β signaling as the critical event in the pathogenesis of aneurysm formation. To determine whether a perturbed elastin lamellar structure arose from induction of transforming growth factor-β-regulated MMPs, we performed gelatin zymography and used a protease-activatable near-infrared fluorescence probe to monitor and quantify MMP upregulation in animals, using various in vivo optical imaging modules and coregistration of the fluorescence signal with CT images of the same animals. Gelatin zymography demonstrated a significant increase in the presence of the active form of MMP-9 in the aortic arch of fibulin-4R/Rmice. In vivo analysis of MMP upregulation using the near-infrared fluorescence probe and subsequent isosurface concentration mapping from reconstructed tomographic images from fibulin-4+/Rand fibulin-4R/Rmice revealed a graded increase in activation of MMPs within the aneurysmal lesions. Conclusions-We aimed to develop molecular imaging procedures for faster, earlier, and easier recognition of aortic aneurysms. We show that in vivo coregistration of MMP activity by noninvasive tomographic imaging methods allows the detection of increased MMP activity, even before the aneurysm has actually formed.

Hyperthermia and thermosensitive liposomes for improved delivery of chemotherapeutic drugs to solid tumors (Article)

2010-08-01T00:00:00Z

Lipid-based nanocarriers or liposomes have been proven successful in the delivery of chemotherapeutic agents and are currently applied clinically in the treatment of various types of cancer. Liposomes offer the advantage of a high drug payload, decreased drug toxicity and enhanced drug accumulation at tumor sites. Increased accumulation is due to the relatively leaky tumor vasculature that allows liposome extravasation. Between different types of tumors and even within one tumor, vascular permeability and thus liposome extravasation may differ greatly. Furthermore, upon accumulation of liposomes in the tumor area, drug bioavailability is not guaranteed. At present, these are the major issues for clinically used liposomal drugs. Mild hyperthermia (HT), the heating of tumor tissue to temperatures of up to 43°C, has been developed in the past decades as an established and efficacious treatment modality in combination with chemo- and radiotherapy. HT can be used to further improve liposomal chemotherapy in two ways: HT is known to increase vascular permeability in solid tumors and may therefore increase levels of liposome accumulation, and thermosensitive liposomes have been developed that can be triggered to release their contents upon hyperthermia. By applying these two strategies, drug delivery to tumors can be strongly enhanced.

Preclinical and Clinical Studies of Peptide Receptor Radionuclide Therapy (Article)

2010-05-01T00:00:00Z

In the 1980s, the111In-labeled somatostatin analog OctreoScan (Covidien, Hazelwood, MO) was developed for imaging of somatostatin receptor subtype 2 (sst2) overexpressing tumors. On the basis of this success, peptide receptor radionuclide therapy (PRRT) was developed using similar somatostatin analogs with different therapeutic radionuclides. Clinical application of PRRT demonstrated impressive results on tumor response, overall survival, and quality of life in patients with gastroenteropancreatic neuroendocrine tumors. The peptides 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), Tyr3-octreotate (DOTATATE) and DOTA, Tyr3-octreotide (DOTATOC) (brand name Onalta), predominantly targeting sst2, have been granted Orphan Drug status by the European Medicines Agency and the US Food and Drug Administration for application in PRRT. Besides somatostatin receptor-targeting peptides, multiple other radiopeptide analogs were developed targeting several other receptors overexpressed on various tumors. Some of these peptide analogs, including cholecystokinin, gastrin, gastrin-releasing peptide, arginine-glycine-aspartate (RGD)-peptides, and glucagon-like peptide 1 analogs appeared very promising in preclinical and clinical imaging and PRRT studies. Although the success of PRRT with radiolabeled somatostatin analogs has been established, there is still room for improvement. The therapeutic window of PRRT could be enlarged by the use of new and improved targeting compounds, of which new antagonists with excellent tumor to background ratios are very promising. Furthermore, locoregional administration, improved healthy tissue protection, and combination treatment can be applied to increase the effectiveness of PRRT. Combination treatment might include cocktails of different peptide analogs of different therapeutic radionuclides and of radiolabeled peptides with chemotherapeutic or radiosensitizing agents. This review summarizes results of PRRT and describes clinical and preclinical studies regarding PRRT optimizing strategies.

Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia (Article)

2010-04-01T00:00:00Z

Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Thermosensitive liposomes (TSL) can release drugs upon heat. This study aims to identify the optimum 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000(DSPE-PEG2000) concentration in stealth TSL to improve content release efficiency under mild hyperthermia (HT). TSL were prepared with DSPE-PEG2000from 1 to 10mol%, around 80nm in size. Quenched carboxyfluorescein (CF) in aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release in dorsal skin flap window chamber models implanted with human BLM melanoma was captured by confocal microscopy. In vitro heat triggered CF release increased with increasing DSPE-PEG2000density. However, 6mol% and higher DSPE-PEG2000caused CF leakage at physiological temperature. TSL with 5mol% DSPE-PEG2000were stable at 37°C, while released 60% CF in 1min and almost 100% CF in 1h at 42°C. In vivo optical intravital imaging showed immediate massive CF release above 41°C. In conclusion, incorporation of 5mol% DSPE-PEG2000optimized stealth TSL content release triggered by HT.

Development of a liposomal delivery system for temperature-triggered release of a tumor targeting agent, Ln(III)-DOTA-phenylboronate (Article)

2010-01-01T00:00:00Z

Liposomes, capable of temperature-triggered content release at the site of interest, can be of great importance for imaging and therapy of tumors. The delivery of imaging agents or therapeutics can be improved by application of liposomes with a gel-to-liquid phase-transition temperature suitable for mild hyperthermia (41-43 °C), and by prolonging their circulation time by incorporation of lipids containing polyethyleneglycol moieties. Still, the rapid wash out of the delivered material from the tumor tissue is a major obstacle for both imaging and therapy. In this study, we developed an optimized temperature sensitive liposomal system to be used with mild hyperthermia: highly stable at physiological temperature and with a sharp transition of the bilayer at 41.5 °C, with subsequent rapid release of entrapped compounds such as calcein or tumor cell-targeting contrast agents. Intravital microscopy on calcein/rhodamine containing liposomes was applied to demonstrate the applicability of this system in vivo. The calcein loaded liposomes were injected iv into nude mice with a human BLM melanoma tumor implanted in a dorsal skin-fold window chamber. Arrival of the liposomes at the tumor site and content release after temperature increase were monitored. The results demonstrated not only accumulation of the liposomes at the tumor site, but also a massive release of calcein after increase of the temperature to 41 °C. The versatility of the thermosensitive liposomes was further demonstrated by encapsulation of a tumor cell-targeting DOTA-phenylboronate conjugate and its release at elevated temperatures. The DOTA ligand in this system is able to chelate a variety of metals suitable for both diagnostic and therapeutic applications, whereas the phenylboronate function is able to target specifically to tumor cells through a covalent binding with sialic acid moieties over-expressed on their surface upon heat-triggered release from the liposomal carrier.

Fluorescence in situ hybridization to monitor the intracellular location and accessibility of plasmid DNA delivered by cationic polymer-based gene carriers (Article)

2009-06-01T00:00:00Z

Information about the intracellular trafficking of exogenous DNA delivered by nonviral gene delivery systems is of major importance for optimization of such gene carriers. We used fluorescence in situ hybridization (FISH) as a tool to visualize polyplex-delivered pDNA inside cells. This avoids the need to directly label DNA inside the polyplexes, which may influence their cellular behavior and fate. Using FISH the introduced plasmid DNA could be detected in the cytosol and nucleus of different cell lines. The FISH probe itself did not interact with cells nor different polymers used for condensing the DNA. We further demonstrate differences in accessibility of polyplex-delivered DNA when different polymers were used for DNA complexation. Therefore, FISH is a valuable tool to detect location and accessibility of exogenous plasmid DNA delivered in the cell by cationic polymers.

Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells (Article)

2007-08-01T00:00:00Z

Purpose. Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. Materials and Methods. The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. Results. Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. Conclusions. These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

Phenylboronate 160Tb complexes for molecular recognition of glycoproteins expressed on tumor cells (Article)

2007-05-18T00:00:00Z

The over-expression of sialic acid on the surface of cancer cells compared with normal ones makes this nine-carbon sugar an attractive biomarker for molecular diagnosis and therapy. Here, we describe a study on the molecular recognition of sialic acid end groups on the surface of human glioma cells by160Tb-DTPA-EN2,160Tb-DTPA-(ENPBA)2and160Tb-DTPA-(PBA)2complexes. The results show Tb-DTPA-(ENPBA)2to be the most efficient targeting agent, due to the electrostatic interaction between its two positively charged ammonium groups and the negatively charged cell surface, which provides an additional stabilization of the covalent binding through the PBA moieties and the sialic acid diol functions. Up to 5.5 nmol Tb/mg protein is taken up by the cells. ICP analysis after incubation experiments with non-radioactive Tb-DTPA-(ENPBA)2Suggests that dissociation of Tb from this complex occurs after its binding to the cell surface. Most likely, most of the free Tb remains adsorbed on the surface of the cells, although internalization of a small amount cannot be excluded. Copyright