Publications

Recent magnetic resonance studies in healthy and cancerous organs have concluded that deuterated metabolites possess highly desirable properties for mapping non-invasively and, as they happen, characterizing glycolysis and other biochemical processes in animals and humans. A promising avenue of this deuterium metabolic imaging (DMI) approach involves looking at the fate of externally administered 2H6,6′-glucose, as it is taken up and metabolized into different products as a function of time. This study employs deuterium magnetic resonance to follow the metabolism of wildtype and preeclamptic pregnant mice models, focusing on maternal and fetoplacental organs over ≈2 h post-injection. 2H6,6′-glucose uptake was observed in the placenta and in specific downstream organs such as the fetal heart and liver. Main metabolic products included 2H3,3′-lactate and 2H-water, which were produced in individual fetoplacental organs with distinct time traces. Glucose uptake in the organs of most preeclamptic animals appeared more elevated than in the control mice (p = 0.02); also higher was the production of 2H-water arising from this glucose. However, the most notable differences arose in the 2H3,3′-lactate concentration, which was ca. two-fold more abundant in the placenta (p = 0.005) and in the fetal (p = 0.01) organs of preeclamptic-like animals, than in control mice. This is consistent with literature reports about hypoxic conditions arising in preeclamptic and growth-restricted pregnancies, which could lead to an enhancement in anaerobic glycolysis. Overall, the present measurements suggest that DMI, a minimally invasive approach, may offer new ways of studying and characterizing health and disease in mammalian pregnancies, including humans.

Vitamin H (biotin) is delivered to the fetus transplacentally by an active biotin-transport mechanism and is critical for fetal development. Our objective was to develop a comprehensive MRI technique for mapping biotin transporter activity in the murine placenta. Visualization of transporter activity can employ MRI’s unique T2*-dependent signal ‘off-switch’, which is triggered by transporter mediated aggregation of biotinylated contrast agent (b-BSA-Gd-DTPA). MRI data were collected from pregnant mice after administration of b-BSA-Gd-DTPA and analyzed using a new sub-voxel biophysical signal model. Validation experiments included competition with native biotin, comparative tests using PET, histology, and ICPMS. MRI signal was governed by binding, aggregation, and clearance of biotin (confirmed by histology). Signal dynamics reflected the placenta’s perfusion pattern modulated by biotin transporter activity and trophoblast mediated retention, and were in congruence with a three-compartment sub-voxel model. Pre-saturation of the transporters with free biotin suppressed b-BSA-Gd-DTPA uptake. The results were confirmed by PET, histology and ICPMS. The presented MRI-based platform allows to track activity of essential molecular transporters in the placenta, reflecting a transporter-mediated uptake, followed by retention and aggregation, and recycling associated with the large b-BSA-Gd-DTPA conjugate. The presented DCE-MRI technique can furthermore be used to map and characterize microstructural compartmentation and transporter activity without exposing the fetus to contrast media.

Background: The extracellular matrix modulates the development of ovarian tumours. Currently evaluation of extracellular matrix in the ovary is limited to histological methods. Magnetic resonance imaging (MRI) and two-photon microscopy (2PM) both enable dynamic visualisation and quantification of fibrosis by endogenous contrasts mechanisms: Magnetization Transfer (MT) MRI and Second Harmonic Generation (SHG) 2PM respectively.Methods: Here we applied MT-MRI protocol for longitudinal imaging of the stroma in orthotopic human ovarian cancer ES-2 xenograft model in CD1 athymic nude mice and for orthotopically implanted ovarian PDX using a MR-compatible imaging window chamber implanted into NSG mice.Results: We observed differences between ECM deposition in ovarian and skin lesions, and heterogenous collagen distribution in ES-2 lesions. An MR-compatible imaging window chamber enabled visual matching between T2 MRI maps of orthotopically implanted PDX grafts and anatomical images of their microenvironment acquired with stereomicroscope and SHG-2PM intravital microscopy of the collagen. Bi-modal MRI / 2PM imaging allowed us to quantify the fibrosis within the same compartments and demonstrated the consistent results across the modalities. Conclusions: This work demonstrates a novel approach for measuring the stromal biomarkers in orthotopic varian tumours in mice, on both macroscopic and microscopic levels.

Pancreatic cancers, both adenocarcinomas and endocrine tumors are characterized by varying levels of aberrant angiogenesis and fibrotic microenvironment. The difficulty to deliver drugs and treat the disease has been attributed in part to the vascular architecture and tissue/ECM density. Here we present longitudinal three-dimensional intravital imaging of vascular and tumor microenvironment remodeling in spontaneous transgenic tumors (RIP1-Tag2 insulinomas) and orthotopically injected tumors (KPC adenocarcinomas). Analysis of the data acquired in insulinomas revealed major differences in tumor blood vessel branching, fraction volume, number of branch points segments, vessel straightness and length compared to the normal tissue. The aggressive adenocarcinoma presented widespread peritumoral vascular remodeling and heterogeneous vascular distribution. Longitudinal imaging was used to acquire sequential vascular remodeling data during tumor progression. This work demonstrates the potential for using a pancreatic intravital imaging window for direct visualization of the tumor heterogenic microenvironments during tumor progression. This article is protected by copyright. All rights reserved.

Angiogenesis, the expansion of the vascular bed, is an important component in remodeling of tissues and organs. Such remodeling is essential for coping with substantial and sustained increase in the demands for supply of oxygen and nutrients and the timely removal of waste products. The vasculature, and its effectiveness in systemic delivery to all parts of the body, regulates the distribution of immune cells and the delivery of therapeutics as well as the dissemination of disease. Therefore, the vascular bed is possibly one of the key organs involved in homeostasis, in health and disease. The critical role of the vasculature in health, and the accessibility to non invasive probing by MRI, renders MRI as a modality of choice for monitoring the vasculature and its adaption to challenges.

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe^-/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received ⁸⁹Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe^-/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. ⁸⁹Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Purpose: To generate magnetic resonance (MR) imaging-derived, oxygen-hemoglobin dissociation curves and to map fetalplacental oxygen-hemoglobin affinity in pregnant mice noninvasively by combining blood oxygen level-dependent (BOLD) T2∗ and oxygen-weighted T1 contrast mechanisms under different respiration challenges. Materials and Methods: All procedures were approved by the Weizmann Institutional Animal Care and Use Committee. Pregnant mice were analyzed with MR imaging at 9.4 T on embryonic days 14.5 (eight dams and 58 fetuses; imprinting control region ICR strain) and 17.5 (21 dams and 158 fetuses) under respiration challenges ranging from hyperoxia to hypoxia (10 levels of oxygenation, 100%-10%; total imaging time, 100 minutes). A shorter protocol with normoxia to hyperoxia was also performed (five levels of oxygenation, 20%-100%; total imaging time, 60 minutes). Fast spin-echo anatomic images were obtained, followed by sequential acquisition of threedimensional gradient-echo T2∗- And T1-weighted images. Automated registration was applied to align regions of interest of the entire placenta, fetal liver, and maternal liver. Results were compared by using a two-tailed unpaired Student t test. R1 and R2∗ values were derived for each tissue. MR imaging-based oxygen-hemoglobin dissociation curves were constructed by nonlinear least square fitting of 1 minus the change in R2∗divided by R2∗at baseline as a function of R1 to a sigmoid-shaped curve. The apparent P50 (oxygen tension at which hemoglobin is 50% saturated) value was derived from the curves, calculated as the R1 scaled value (x) at which the change in R2∗ divided by R2∗at baseline scaled (y) equals 0.5. Results: The apparent P50 values were significantly lower in fetal liver than in maternal liver for both gestation stages (day 14.5: 21% ± 5 [P = .04] and day 17.5: 41% ± 7 [P , .0001]). The placenta showed a reduction of 18% ± 4 in mean apparent P50 values from day 14.5 to day 17.5 (P = .003). Reproduction of the MR imaging-based oxygenhemoglobin dissociation curves with a shorter protocol that excluded the hypoxic periods was demonstrated. Conclusion: MR imaging-based oxygen-hemoglobin dissociation curves and oxygen-hemoglobin affinity information were derived for pregnant mice by using 9.4-T MR imaging, which suggests a potential to overcome the need for direct sampling of fetal or maternal blood.

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r₂ relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R₂ relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.

The goal of this work was to study the efficacy of the positron emission tomography (PET) tracers 2-deoxy-2-[F-18]fluoro-D-glucose ([F-18]FDG) and Cu-64-diacetyl-bis(N4-methylthiosemicarbazone) ([Cu-64]ATSM) and in monitoring placental and fetal functional response to acute hyperoxia in late-term pregnant mice subjected to experimentally induced chronic hypoxia. E15 mice were maintained at 12% inspired oxygen for 72 hours and then imaged during oxygen inhalation with either [F-18]FDG to monitor nutrient transport or Cu-64-ATSM to establish the presence of hypoxia. Computed tomography (CT) with contrast allowed clear visualization of both placentas and fetuses. The average ratio of fetal to placental [F-18]FDG uptake was 0.45 +/- 0.1 for the hypoxic animals and 0.55 +/- 0.1 for the normoxic animals, demonstrating a significant decrease (p =.0002) in placental function in dams exposed to chronic hypoxic conditions. Hypoxic placentas and fetuses retained more Cu-64-ATSM compared to normoxic placentas and fetuses. Herein we report first-in-mouse PET imaging of fetuses employing both tracers [F-18]FDG (metabolism) and Cu-64-ATSM (hypoxia). [F-18]FDG PET/CT imaging allowed clear visualization of placental-fetal structures and supported quantification of tracer uptake, making this a sensitive tool for monitoring placental function in preclinical rodent models. These measurements illustrate the potentially irreversible damage generated by chronic exposure to hypoxia, which cannot be corrected by acute exposure to hyperoxia.

The placenta performs a wide range of physiological functions; insufficiencies in these functions may result in a variety of severe prenatal and postnatal syndromes with long-term negative impacts on human adult health. Recent advances in magnetic resonance imaging (MRI) studies of placental function, in both animal models and humans, have contributed significantly to our understanding of placental structure, blood flow, oxygenation status, and metabolic profile, and have provided important insights into pregnancy complications.

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43 ^del/del). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43^del/del oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43^del/del oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF.

Objective To explore the role of Akt1, a principle modulator of angiogenesis, in ovarian graft reception and to investigate whether Akt1 deficiency can alter ovarian graft reception. Design Experimental mouse model. Setting Research institute. Animal(s) Donors: Akt1 knockout (Akt1^-/-) and wild types (Akt1^+/+) mice. Recipients: CD-1 nude immune deficient female mice. Intervention(s) Ovaries from Akt1^-/- and Akt1^+/+ mice transplanted in the biceps femoris muscle of immunocompromised CD-1 mice, and ovarian graft viability, perfusion, and revascularization explored in vivo by magnetic resonance imaging (MRI). Main Outcome Measure(s) Vascular density and permeability of newly formed graft blood vessels quantified by dynamic contrast-enhanced MRI 7, 14, 30, and 60 days after grafting as indicators for angiogenesis and reestablishment of blood perfusion. Result(s) The Akt1^-/- ovarian grafts showed a gradual decrease in angiogenic response with time after transplantation, ultimately leading to complete or near-complete graft destruction coinciding with massive follicular loss. Sixty days after transplantation, the mean blood volume fraction (fBV) and vessel permeability (PS) were statistically significantly lower in Akt1^-/- transplants compared with Akt1^+/+. Conclusion(s) Akt1 is essential for ovarian graft reception. However, surprisingly the impact of Akt1 deficiency was most profound not in the early stages of angiogenesis but rather in long-term survival of the graft.

Cancer cells rely on extensive support from the stroma in order to survive, proliferate and invade. The tumor stroma is thus an important potential target for anti-cancer therapy. Typical changes in the stroma include a shift from the quiescence promoting-antiangiogenic extracellular matrix to a provisional matrix that promotes invasion and angiogenesis. These changes in the extracellular matrix are induced by changes in the secretion of extracellular matrix proteins and glucose amino glycans, extravasation of plasma proteins from hyperpermeable vessels and release of matrix modifying enzymes resulting in cleavage and cross-linking of matrix macromolecules. These in turn alter the rigidity of the matrix and the exposure and release of cytokines. Changes in matrix rigidity and vessel permeability affect drug delivery and mediate resistance to cytotoxic therapy. These stroma changes are brought about not only by the cancer cells, but also through the action of many cell types that are recruited by tumors including immune cells, fibroblasts and endothelial cells. Within the tumor, these normal host cells are activated resulting in loss of inhibitory and induction of cancer promoting activities. Key to the development of stroma-targeted therapies, selective biomarkers were developed for specific imaging of key aspects of the tumor stroma.

Even though congenital heart disease is the most prevalent malformation, little is known about how mutations affect cardiovascular function during development. Akt1 is a crucial intracellular signaling molecule, affecting cell survival, proliferation, and metabolism. The aim of this study was to determine the role of Akt1 on prenatal cardiac development. In utero echocardiography was performed in fetal wild-type, heterozygous, and Akt1-deficient mice. The same fetal hearts were imaged using ex vivo micro-computed tomography (lCT) and histology. Neonatal hearts were imaged by in vivo magnetic resonance imaging. Additional ex vivo neonatal hearts were analyzed using histology and real-time PCR of all three groups. In utero echocardiography revealed abnormal blood flow patterns at the mitral valve and reduced contractile function of Akt1 null fetuses, while ex vivo μCT and histology unraveled structural alterations such as dilated cardiomyopathy and ventricular septum defects in these fetuses. Further histological analysis showed reduced myocardial capillaries and coronary vessels in Akt1 null fetuses. At neonatal age, Akt1-deficient mice exhibited reduced survival with reduced endothelial cell density in the myocardium and attenuated cardiac expression of vascular endothelial growth factor A and collagen Iα1. To conclude, this study revealed a central role of Akt1 in fetal cardiac function and myocardial angiogenesis inducing fetal cardiomyopathy and reduced neonatal survival. This study links a specific physiological phenotype with a defined genotype, namely Akt1 deficiency, in an attempt to pinpoint intrinsic causes of fetal cardiomyopathies.

Purpose: To quantitatively monitor the dynamic perivascular recruitment of ferritin heavy chain (FHC)-overexpressing fibroblasts to ovarian carcinoma xenografts by using R2 mapping and biexponential magnetic resonance (MR) relaxometry. Materials and Methods: In vivo studies of female mice were approved by the institutional animal care and use committee. In vitro analysis included MR-based R2 relaxation measurements of monkey kidney cell line (CV1) fibroblasts that overexpress FHC, followed by inductively coupled plasma mass spectrometry to assess cellular iron content. For in vivo analysis, CV1-FHC fibroblasts were either mixed with fluorescent human ovarian carcinoma cells before subcutaneous implantation (coinjection) or injected intraperitoneally 4 days after the cancer cells were injected (remote recruitment). Dynamic changes in tumor R2 were used to derive CV1-FHC cell fraction in both models. In coinjection tumors, dynamic contrast material-enhanced MR imaging was used to measure tumor fractional blood volume. Whole-body fluorescence imaging and immunohistochemical staining were performed to validate MR results. One-way repeated measures analysis of variance was used to assess MR and fluorescence imaging results and tumor volume, and one-way analysis of variance was used to assess spectrometric results, fractional blood volume, and immunohistochemical evaluation. Results: CV1-FHC fibroblasts (vs CV1 fibroblasts) showed enhanced iron uptake (1.8 mmol ± 0.5 × 10^-8 vs 0.9 mmol ± 0.5 × 10^-8; P -8 vs 0.5 mmol ± 0.5 × 10^-8, P -3 for CV1, 2.3 AU ± 0.3 × 10^-3 for CV1-FHC, 2.9 ± 0.3 × 10^-3 for CV1-FHC-ferric citrate). Dynamic changes in CV1-FHC cell fraction determined at MR relaxometry in both models were confirmed at immunohistochemical analysis. Conclusion: FHC overexpression, when combined with R2 mapping and MR relaxometry, enabled in vivo detection of the dynamic recruitment of exogenously administered fibroblasts to the vasculature of solid tumors.

Inflammatory bowel disease is a chronic inflammatory disorder of the gastrointestinal tract associated with alterations and dysfunction of the intestinal microvasculature. The goal of this work was to develop a preclinical protocol for quantitative functional characterization of the colonic microvasculature in a murine colitis model. Experimental colitis was induced in mice by addition of dextran sodium sulfate to the drinking water. Histopathologic analysis revealed severe multifocal colitis. Dynamics of intravenously injected macromolecular dextran-FITC and biotin-BSA-GdDTPA in the colonic microvasculature were imaged using fluorescent confocal endomicroscopy and MRI (9.4 T), respectively. Both MRI and fluorescent confocal endomicroscopy revealed a substantial increase in the permeability of the colonic microvasculature associated with colitis, resulting in extravascular accumulation of the macromolecular contrast agent in the lumen of the colon. MRI data were validated by immunohistochemical staining of the contrast agent and leakage of fluorescently labeled BSA-FAM coinjected with the MRI contrast agent. Leakage of plasma proteins and deposition of a provisional matrix can support inflammation and stimulate remodeling of the colonic vasculature. Thus, the plasma protein leakage from the colonic microvasculature at the focal inflammatory patches could be quantified by MRI, providing a biomarker for assessment of disease progression. Magn Reson Med, 2011. © 2011 Wiley-Liss, Inc.

Purpose: The purpose of this study was to develop a tool for functional phenotyping of the maternal circulation in the mouse placenta. Procedures: In utero macromolecular dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed on embryonic day 10.5 (E10.5), E13.5, and E18.5. Fluorescence analysis was also used for validation of the results. Results: The initial rate of contrast enhancement revealed an increased maternal blood volume fraction as the pregnancy progressed. Serial imaging of E10.5 and E13.5 placentas revealed a loss of contrast enhancement due to phagocytic uptake. A key application of macromolecular DCE-MRI would be to follow mouse pregnancies during fetal and placental manipulation including embryo transfer, tetraploid complementation, and fetal resorptions. We were able to resolve strain differences in ICR outbred mice carrying both ICR and C57Bl/6J embryos and to differentiate in utero resorptions from functional placentas. Conclusions: Our results highlight the importance of the functional in utero analysis of placental vascularization in physiological phenotyping of transgenic mice and suggest MRI, particularly macromolecular DCE-MRI, as a non-invasive tool for the analysis of the placenta.

The importance of placental circulation is exemplified by the correlation of placental size and blood flow with fetal weight and survival during normal and compromised human pregnancies in such conditions as preeclampsia and intrauterine growth restriction (IUGR). Using noninvasive magnetic resonance imaging, we evaluated the role of PKBalpha/AKT1, a major mediator of angiogenesis, on placental vascular function. PKBalpha/AKT1 deficiency reduced maternal blood volume fraction without affecting the integrity of the fetomaternal blood barrier. In addition to angiogenesis, PKBalpha/AKT1 regulates additional processes related to survival and growth. In accordance with reports in adult mice, we demonstrated a role for PKBalpha/AKT1 in regulating chondrocyte organization in fetal long bones. Using tetraploid complementation experiments with PKBalpha/AKT1-expressing placentas, we found that although placental PKBalpha/AKT1 restored fetal survival, fetal PKBalpha/AKT1 regulated fetal size, because tetraploid complementation did not prevent intrauterine growth retardation. Histological examination of rescued fetuses showed reduced liver blood vessel and renal glomeruli capillary density in PKBalpha/Akt1 null fetuses, both of which were restored by tetraploid complementation. However, bone development was still impaired in tetraploid-rescued PKBalpha/Akt1 null fetuses. Although PKBalpha/AKT1-expressing placentas restored chondrocyte cell number in the hypertrophic layer of humeri, fetal PKBalpha/AKT1 was found to be necessary for chondrocyte columnar organization. Remarkably, a dose-dependent phenotype was exhibited for PKBalpha/AKT1 when examining PKBalpha/Akt1 heterozygous fetuses as well as those complemented by tetraploid placentas. The differential role of PKBalpha/AKT1 on mouse fetal survival and growth may shed light on its roles in human IUGR.

Biological imaging studies of fetal development are frequently conducted on small laboratory animals, which offer the advantages of rapid reproductive cycle and multiparity. The first section of this chapter will screen the most widely used animal models. Animal models aiming to study human physiology or disease by noninvasive imaging should exhibit along with genetic, anatomical, and physiological similarities to humans, also the ability to provide information using available imaging modalities. Many developmental studies utilized the rapid reproduction, easy access, and optical clarity of developing avian and fish embryos for high-resolution fluorescence microscopy, while studies of mammals were frequently limited to ex vivo imaging. However, over the last years, new imaging tools allow in vivo monitoring of development also in the mouse, which is the most common mammalian model for the study of development, genetics, immune response, pathology, neurology, and cellular mechanisms of action.

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

The dynamic light scattering imaging (DLSI) system combined with the conventional fluorescence intravital microscope (FIM) has been applied for the examination of blood and lymph vessels in the mouse ear in vivo. While the CCD camera can be shared by both techniques the combined application of DLSI and FIM allows rapid switching between the modalities. In current study temporal speckles fluctuations are used for rendering blood vessels structure and monitoring blood perfusion with the higher spatial resolution, whereas FIM provides the images of lymphatic vessels. The results clearly demonstrate that combined application of DLSI and FIM approaches provides synchronic in vivo images of blood and lymph vessels with higher contrast and specificity. The use of this new dual-modal diagnostic system is particularly important and has a great potential to significantly expand the capabilities of vascular diagnostics providing synchronic in vivo images of blood and lymph vessels.

PKBalpha/Akt1, a protein kinase, is a major mediator of angiogenic signaling. The purpose of this study was to determine the role of PKBalpha/Akt1 in bone vascularization and development. For that aim, macromolecular dynamic contrast enhanced MRI was applied to examine in vivo vascular changes in long bones of 40-day-old growing PKBalpha/Akt1-deficient, heterozygous, and wild-type mice. Ex vivo μMRI and μCT were applied to monitor the impact of PKBalpha/Akt1 gene dosage on trabecular bone formation during endochondral bone growth. PKBalpha/Akt1-deficient mice and, remarkably, also heterozygous mice showed significantly reduced blood volume fraction in the humerus compared to wild-type mice. Moreover, PKBalpha/Akt1-deficient mice showed a more severe vascular deficiency with reduced permeability. μCT and μMRI of trabeculae revealed impaired bone formation in both PKBalpha/Akt1-deficient and heterozygous mice, whereas cortical bone parameters were only reduced in PKBalpha/Akt1-deficient mice. Reduction of metaphyseal blood vessel invasion, concomitant with aberrant trabeculae and shorter long bones, demonstrates a gene-dose-dependent role for PKBalpha/Akt1 in regulation of overall size and endochondral bone growth. MRI proved to provide high sensitivity for in vivo detection of subtle gene dose effects leading to impaired bone vascularity and for uncovering changes in trabecular bone.

Purpose: To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo. Materials and Methods: All experiments were approved by the governmental review committee on animal care. USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test. Results: HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and α _vβ₃ integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO. Conclusion: In glioma cells with unstable intercellular contacts, inhibition of α_vβ₃ integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes.

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)-based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/ CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSAGdDTPA/ CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSAGdDTPA/ CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSAGdDTPA/ CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells.

Keywords: Developmental Biology

Noninvasive molecular imaging of dynamic processes has benefited tremendously from the use of reporter genes. These genes encode for proteins that emit light, bind radiolabeled probes, or, as covered in this review, modulate MRI contrast. Reporter genes play a pivotal role in monitoring cell trafficking, gene replacement therapy, protein-protein interactions, neuronal plasticity, and embryonic development. Several strategies exist for generating MRI contrast: using enzyme-catalyzed chemical modification of metal-based contrast agents or (phosphorus) metabolites, iron-binding and iron-storage proteins to accumulate iron as a contrast agent, and artificial proteins for imaging based on chemical exchange saturation transfer. MRI reporter genes have the advantage that the specific signal can be coregistered with soft-tissue anatomy and functional tissue information and have, therefore, become an active and growing area of scientific interest.

Tumor-associated stroma, in general, and tumor fibroblasts and myofibroblasts, in particular, play a role in tumor progression. We previously reported that myofibroblast infiltration into implanted ovarian carcinoma spheroids marked the exit of tumors from dormancy and that these cells contributed to vascular stabilization in ovarian tumors by expression of angiopoietin-1 and angiopoietin-2. Ex vivo labeling of fibroblasts with either magnetic resonance or optical probes rendered them detectable for in vivo imaging. Thus, magnetic resonance imaging (MRI) follow-up was feasible by biotin-bovine serum albumin-gadolinium diet-hylenetriaminepentaacetic acid or iron oxide particles, whereas labeling with near-IR and fluorescent vital stains enabled in vivo visualization by near-IR imaging and two-photon microscopy. Using this approach, we show here that prelabeled fibroblasts given i.p. to CD-1 nude mice can be followed in vivo by MRI and optical imaging over several days, revealing their extensive recruitment into the stroma of remote s.c. MLS human epithelial ovarian carcinoma tumors. Two-photon microscopy revealed the alignment of these invading fibroblasts in the outer rim of the tumor, colocalizing with the angiogenic neovasculature. Such angiogenic vessels remained confined to the stroma tracks within the tumor and did not penetrate the tumor nodules. These results provide dynamic evidence for the role of tumor fibroblasts in maintenance of functional tumor vasculature and offer means for image-guided targeting of these abundant stroma cells to the tumor as a possible mechanism for cellular cancer therapy.

Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R ₂) of water. Transgene-dependent changes in R₂ were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.

One of the attractions of molecular imaging using 'smart' bioactive contrast agents is the ability to provide non-invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA-GdDTPA-beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first-order kinetic analysis of the time-dependent increase in R(2) as a result of hyaluronidase activity. The changes in R(2) and the measured relaxivity of intact HA-GdDTPA-beads (r(2B)) and HA-GdDTPA fragments (r(2D)) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA-GdDTPA-beads as the consequence of the release of HA-GdDTPA fragments. The rate of dissociation of HA-GdDTPA from the beads showed typical bell-shaped temperature dependence between 7 and 36 degrees C with peak activity at 25 degrees C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA-GdDTPA-beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases.

Interactions between the tumor stromal compartment and cancer cells play an important role in the spread of cancer. In this study, we have used noninvasive in vivo magnetic resonance imaging (MRI) of two human breast cancer models with significantly different invasiveness, to quantify and understand the role of interstitial fluid transport, lymphatic-convective drain, and vascularization in the regional spread of breast cancer to the axillary lymph nodes. Quantitative fluorescence microscopy was done to morphometrically characterize lymphatic vessels in these tumors. Significant differences in vascular and extravascular transport variables as well as in lymphatic vessel morphology were detected between the two breast cancer models, which also exhibited significant differences in lymph node and lung metastasis. These data are consistent with a role of lymphatic drain in lymph node metastasis and suggest that increased lymph node metastasis may occur due to a combination of increased invasiveness, and reduced extracellular matrix integrity allowing increased pathways of least resistance for the transport of extravascular fluid, as well as tumor cells. It is also possible that lymph node metastasis occurred via the cancer cell-bearing tumoral lymphatic vessels. The congestion of these tumoral lymphatics with cancer cells may have restricted the entry and transport of macromolecules.

Hyaluronan, a high molecular weight, negatively charged polysaccharide, is a major constituent of the extracellular matrix. High molecular weight hyaluronan is antiangiogenic, but its degradation by hyaluronidase generates proangiogenic breakdown products. Thus, by expression of hyaluronidase, cancer cells can tilt the angiogenic balance of their microenvironment. Indeed, hyaluronidase-mediated breakdown of hyaluronan correlates with aggressiveness and invasiveness of ovarian cancer metastasis and with tumor angiogenesis. The goal of this work was to develop a novel smart contrast material for detection of hyaluronidase activity by magnetic resonance imaging (MRI). Gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) covalently linked to hyaluronan on the surface of agarose beads showed attenuated relaxivity. Hyaluronidase, either purified from bovine testes or secreted by ES-2 and OVCAR-3 human epithelial ovarian carcinoma cells, activated the hyaluronan-GdDTPA-beads by rapidly altering the R₁ and R₂ relaxation rates. The change in relaxation rates was consistent with the different levels of biologically active hyaluronidase secreted by those cells. Hyaluronan-GdDTPA-beads were further used for demonstration of MRI detection of hyaluronidase activity in the proximity of s.c. ES-2 ovarian carcinoma tumors in nude mice. Thus, hyaluronan-GdDTPA-beads could allow noninvasive molecular imaging of hyaluronidase-mediated tilt of the peritumor angiogenic balance.

Heparanase expression has been linked to increased tumor invasion, metastasis, and angiogenesis and with poor prognosis. The aim of the study was to monitor the effect of heparanase expression on lymph node metastasis, in heparanase-overexpressing subcutaneous Eb mouse T-lymphoma tumors, and their draining lymph node. Dynamic contrast-enhanced magnetic resonance imaging (MRI) using biotin-BSA-GdDTPA-FAM/ROX was applied for analysis of blood volume, vascular permeability, and interstitial convection, and for detection of very early stages of such metastatic dissemination. Eb tumors increased extravasation, interstitial convection, and lymphatic drain of the contrast material. Interstitial flow directions were mapped by showing radial outflow interrupted in some tumors by directional flow toward the popliteal lymph node. Heparanase expression significantly increased contrast enhancement of the popliteal lymph node but not of the primary tumor. Changes in MR contrast enhancement preceded the formation of pathologically detectable metastases, and were detectable when only a few enhanced green fluorescent protein (EGFP)-expressing Eb cells were found near and within the nodes. These results demonstrate very early, heparanase-dependent vascular changes in lymph nodes that were visible by MRI following administration of biotin-BSA-GdDTPA-FAM/ROX, and can be used for studying the initial stages of lymph node infiltration.

MLS ovarian epithelial carcinoma multicellular spheroids xenografted subcutaneously in CD-I nude mice displayed growth delay, or dormancy, of up to 52 days. In the study reported here, implanted MLS spheroids were used for testing the role of angiogenesis and vascular maturation in triggering the initiation of tumor progression. The kinetics and impact of neovascular maturation and functionality, in dormancy, and growth of MLS spheroid xenografts were studied noninvasively by BOLD contrast MRI. MR data were supported by histologic staining for biotinylated albumin as a blood pool marker and alpha-smooth muscle actin (alpha-SMA) as marker for perivascular mural cells. Although the tumor periphery showed higher levels of total and mature vasculature than normal skin, the fraction of mature out of the total vessels as detected by MRI vascular maturation index (VMI_MRI) was significantly lower in the tumor both before and after tumor exit from dormancy. The neovasculature induced by the implanted spheroid was unstable and showed cycles of vessel growth and regression. Surprisingly, this instability was not restricted to the immature vessels, but rather included also regression of mature vessels. During dormancy, neovasculature was predominantly peripheral with no infiltration into the implanted spheroid. Infiltration of alpha-SMA positive stroma cells into the spheroid was associated with functional vascularization and tumor growth. Thus, stroma infiltration and vascular maturation are an important checkpoint linking the angiogenic switch with initiation of tumor progression.

Halofuginone has recently been shown to inhibit tumor progression of various types of cancers. The antitumoral effect was associated with decreased tumor angiogenesis rather than a direct cytostatic effect on the tumor cells. The antiangiogenic action of the drug could be related to its inhibition of collagen type I synthesis, inhibition of matrix metalloproteinases (MMPs), or via both mechanisms because both collagen synthesis and MMP activity have been shown to be involved in angiogenesis. Vascular endothelial growth factor (VEGF), in addition to its effect on endothelial cell proliferation, has been shown to be a potent inducer of MMP expression. Because von Hippel-Lindau (VHL)-associated tumors express high levels of VEGF, it was of interest to ascertain the potential usefulness of halofuginone for treatment of these tumors. Pheochromocytoma tissue fragments obtained at surgery from a VHL type 2a patient were propagated s.c. in male BALB/c v/v (nude) mice. For experiments, 2-3-mm tumor fragments were transplanted secondarily s.c. to nude mice. Two treatment groups received halofuginone in standard lab chow at 3 and 5 ppm; control animals received regular chow. All groups were followed for 6 weeks after transplantation. A marked and significant diminution of tumor size and weight was observed in the drug-treated animals (>90% reduction of mean tumor volume for both the 3 and 5 ppm groups). In vivo magnetic resonance imaging analysis of tumors in halofuginone-treated animals showed a significant reduction of vascular functionality. Immunohistoehemical studies revealed decreased collagen type I levels and vascular density in treated tumors and gelatinase assays of tumor extracts revealed a reduction of MMP-2 and MMP-9 activity in halofuginone-treated cells. Taken together, our data indicate that therapy directed at blocking MMP activity (presumably related to excessive VEGF expression in VHL) and reduction of type I collagen deposition curtails angiogenesis and thereby presumably tumor growth in this model system.

Cancer patients, treated by either chemo- or radiotherapy, frequently suffer from ovarian failure and infertility. One of the new emerging techniques to preserve reproductive potential of such patients is cryopreservation of ovarian fragments prior to treatment and their retransplantation after healing. A major obstacle in survival of the ovarian implants is vascular failure, which leads to tissue necrosis. In order to investigate the role of angiogenesis in implant preservation, we used a xenograft model in which rat ovaries were transplanted into immunodeficient mice. Graft reception and maintenance were monitored by magnetic resonance imaging (MRI) and histology. Two transplantation sites were explored, i.e., subcutaneous and intramuscular. Comparison between these two transplantation sites revealed the importance of vascular smooth muscle cells and pericytes in sustaining vascular and tissue integrity. Histological examination of the grafts, at different time points and sizes, revealed that loss of perivascular cells preceded damage to endothelial cells and was closely correlated with loss of follicular and oocyte integrity. Intramuscular implantation provided better maintenance of implant perivascular cells relative to subcutaneous implantation. Accordingly, follicular integrity was superior in the intramuscular implants and the number of damaged follicles was significantly lower compared with the subcutaneous transplantation site. These results suggest that improving ovarian implant maintenance should be directed toward preservation of perivascular support.

Magnetic resonance imaging (MRI) was employed for non-invasive analysis of vascular remodeling during follicular maturation in the PMSG/hCG rat ovary model. Changes in water diffusion and in perfusion led us to suggest that hypoxic stress may be a component in the regulation of angiogenesis in the growing follicle. However, in contrast with solid tumors of similar size, the spatial and temporal pattern of expression of vascular endothelial growth factor (VEGF), did not match the angiogenic response. The mismatch could be explained by the role of hyaluronan as a high molecular weight suppressor of angiogenesis maintaining an avascular follicular antrum.

Objective. The goal of this work was to evaluate the involvement of gonadotropins in the regulation of adhesion of human epithelial ovarian carcinoma. We studied two pathways that were previously implicated in the metastatic implantation of ovarian carcinoma to the peritoneum, namely hyaluronan-CD44 and RGD-integrin mediated adhesion. Methods. Two cell lines derived from human epithelial ovarian carcinoma (MLS and OC238) were stimulated with luteinizing hormone (LH) and/or follicle stimulating hormone (FSH). Expression of CD44 was evaluated by Western blotting. Expression of αv-integrins was studied by RT-PCR and Northern blot. Integrin and CD44 mediated adhesion of the cells was analyzed using culture plates coated either with a thrombin derived RGD containing peptide or fibronectin for integrin mediated adhesion or with hyaluronan for CD44 mediated adhesion. Results. MLS cells stimulated with either LH or FSH showed increased adhesion to culture plates coated with hyaluronan, as well as to culture plates coated with fibronectin or with a thrombin derived RGD containing peptide. In these cells, gonadotropin stimulation led to induced expression of the integrin subunit α_v and CD44, the cell surface hyaluronan receptor. On the other hand, OC238 cells showed no expression of the integrin subunit α_v and no hormonal effect on the expression of CD44. Accordingly, adhesion of OC238 cells on either RGD or CD44 was not affected by hormonal stimulation. Conclusions. Elevated levels of gonadotropins may in some cases facilitate peritoneal metastatic dissemination of ovarian cancer by increasing cell adhesion, the first essential step in the invasion process.

Spinal cord injury and its devastating consequences are the subject of intensive research aimed at reversing or at least minimizing functional loss. Research efforts focus on either attenuating the post-injury spread of damage (secondary degeneration) or inducing some regeneration. In most of these studies, as well as in clinical situations, evaluation of the state of the injured spinal cord poses a serious difficulty. To address this problem, we carried out a diffusion-weighted MRI experiment and developed an objective routine for quantifying anisotropy in injured rat spinal cords. Rats were subjected to a contusive injury of the spinal cord caused by a controlled weight drop. Untreated control rats were compared with rats treated with T cells specific to the central nervous system self-antigen myelin basic protein, a form of therapy recently shown to be neuroprotective. After the rats were killed their excised spinal cords were fixed in formalin and imaged by multislice spin echo MRI, using two orthogonal diffusion gradients. Apparent diffusion coefficient (ADC) values and anisotropy ratio (AI) maps were extracted on a pixel-by-pixel basis. The calculated sum of AI values (SAI) for each slice was defined as a parameter representing the total amount of anisotropy. The mean-AI and SAI values increased gradually with the distance from the site of the lesion. At the site itself, the mean-AI and SAI values were significantly higher in the spinal cords of the treated animals than in the controls (P = 0.047, P = 0.028, respectively). These values were consistent with the score of functional locomotion. The difference was also manifested in the AI maps, which revealed well-organized neural structure in the treated rats but not in the controls. The SAI values, AI histograms, and AI maps proved to be useful parameters for quantifying injury and recovery in an injured spinal cord. These results encourage the development of diffusion anisotropy MRI as a helpful approach for quantifying the extent of secondary degeneration and measuring recovery after spinal cord injury.

Bold contrast MRI was applied for mapping vascular maturation in tumor- and wound-induced skin angiogenesis using the response of mature vessels to hypercapnia (inhalation of air vs. air 5% CO₂) and the response of all vessels to hyperoxia (air 5% CO₂ vs. oxygen 5% CO₂ (carbogen)). MRI signal enhancement with hypercapnia was reduced in centered vs. linear phase encoding, suggesting increased blood flow. However, intravital microscopy demonstrated constriction of arterioles and reduced flux and density of red blood cells in mature capillaries with hypercapnia, with no change in the diameter of wound-induced neovasculature. The discrepancy in flow between MRI and intravital microscopy is consistent with increased plasma flow and reduced hematocrit. Hyperoxia resulted in increased blood oxygenation and constriction of all vessels. These results provide a hemodynamic explanation for the selective registration of MRI response to hypercapnia with mature vessels and the response to hyperoxia with total vascular function.

A solid tumor presents a unique challenge as a system in which the dynamics of the relationship between vascularization, the physiological environment and metabolism are continually changing with growth and following treatment. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) studies have demonstrated quantifiable linkages between the physiological environment, angiogenesis, vascularization and metabolism of tumors. The dynamics between these parameters continually change with tumor aggressiveness, tumor growth and during therapy and each of these can be monitored longitudinally, quantitatively and non-invasively with MRI and MRS. An important aspect of MRI and MRS studies is that techniques and findings are easily translated between systems. Hence, pre-clinical studies using cultured cells or experimental animals have a high connectivity to potential clinical utility. In the following review, leaders in the field of MR studies of basic tumor physiology using pre-clinical models have contributed individual sections according to their expertise and outlook. The following review is a cogent and timely overview of the current capabilities and state-of-the-art of MRI and MRS as applied to experimental cancers. A companion review deals with the application of MR methods to anticancer therapy.

Autoimmune T cells against central nervous system myelin associated peptide reduce the spread of damage and promote recovery in injured rat spinal cord, findings that might lead to neuroprotective cell therapy without risk of autoimmune disease.

One of the hallmarks of tumor neovasculature is the prevalence of immature vessels manifested by the low degree of recruitment of vascular mural cells such as pericytes and smooth muscle cells. This difference in the architecture of the vascular bed provides an important therapeutic window for inflicting tumor-selective vascular damage. Here we demonstrate the application of gradient echo magnetic resonance imaging (MRI) for noninvasive in vivo mapping of vascular maturation, manifested by the ability of mature vessels to dilate in response to elevated levels of CO₂. Histological α- actin staining showed a match between dilating vessels detected by MRI and vessels coated with smooth muscle cells. Switchable, vascular endothelial growth factor (VEGF)-overexpressing tumors (C6pTET-VEGF rat glioma s.c. tumors in nude mice) displayed high vascular function and significant vascular damage upon VEGF withdrawal. However, damage was restricted to nondilating vessels, whereas mature dilating tumor vessels were resistant to VEGF withdrawal. Thus, MRI provides in vivo visualization of vascular maturity and prognosis of vascular obliteration induced by VEGF withdrawal.

Pulsed arterial spin labeling was used for mapping ovarian perfusion and measurement of blood velocity in the ovarian artery. Arterial blood was tagged upstream by pulsed slice selective saturation, and saturation transfer due to perfusion was monitored within the rat ovary. The velocity of arterial blood was determined from the dependence of the saturation transfer on the thickness of the saturation slice and the delay between successive saturation pulses. This method allows for determination of arterial velocity, even when the artery itself is not identified in the images. The arterial velocity of blood to the ovary was 3.6 ± 0.6 cm · s^-1. The mean ovarian perfusion was 8.7 ± 3.5 ml · min^-1 · g^-1 during the surge of luteinizing hormone and 5.9 ± 3.0 ml · min^-1 · g^-1 during the luteal phase. Arterial labeling can thus be used for following vascular remodeling and angiogenesis during the ovarian cycle by MRI.

The goal of this study was to develop an experimental method for noninvasive analysis of angiogenesis, namely the sprouting of capillaries from existing blood vessels. Angiogenesis was assayed in the subcutaneous vasculature of nude mice in a region of 3 x 3 cm that included in its center a defined angiogenic stimulus. Angiogenic stimuli included agarose beads containing angiogenic growth factors, multicellular tumor spheroids, and dental incisions. Highly significant correlation (r = 0.905, P = 0.0001) was found between the apparent vessel density determined by gradient echo MRI and the density of blood-containing vessels determined postmortem. The functionality of the neovasculature was demonstrated in mice breathing alternatingly carbogen or 95% air/5% CO₂. Large signal enhancement with carbogen breathing corresponded to regions of high vessel density. The assay reported here can be applied for the study of dermal wound healing, primary vascularization of subcuteneous implants, and for measuring the activity of angiogenic and antiangiogenic agents.

Keywords: Cardiac & Cardiovascular Systems; Pharmacology & Pharmacy; Peripheral Vascular Disease

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

Magnetic resonance imaging has been used to follow noninvasively tumor neovascularization and tumor growth in a model system of multicellular C6 rat glioma spheroids implanted s.c. in nude mice. By positioning a single spheroid approximately 1 cm from the site of incision both the vascularization of the tumor and the wound healing processes were spatially separated and could be simultaneously followed. The model proposed here provides defined initial conditions of tumor geometry and cell proliferative status and separation of initial tumor growth from neovascularization. Magnetic susceptibility relaxation provided an intrinsic marker for blood containing vessels. The implanted spheroid induced vessel growth within 4 days after implantation that was geometrically oriented toward the spheroid and distinct from wound healing at the site of incision. Volume measurements showed a corresponding 4-day lag in growth followed by Gompertz progression. Sham implantation of agarose beads of similar diameter showed no induction of vessel growth, ruling out a direct effect of wound healing. The new vessels penetrating the tumor were highly permeable to the contrast reagent gadolinium-diethylenetri-aminepentaacetic acid. This permeability may be due to the action of vascular endothelial growth factor, a major angiogenic growth factor in this system, and a potent permeability factor.

Nuclear magnetic resonance microimaging measurements of the self-diffusion coefficient of water in large (> 2 mm) EMT-6 multicellular spheroids were performed in order to elucidate diffusion mechanisms in tumors. Pulsed gradient spin echo-imaging methods were developed for measuring diffusion in an intravoxel multicompartment system. The self-diffusion coefficient (at 22-degrees-C) for water in the medium (D(m)) consisted of only a single diffusion compartment [D(m) = 1.99 +/- 0.03 (SE) x 10(-5) cm2/s]. Similarly, the spheroid necrotic center showed a single water diffusion compartment with a self-diffusion coefficient (D(c)) significantly lower than that of the medium (D(c) = 1.54 +/- 0.05 X 10(-5) cm2/s). The spheroid viable rim region showed two distinct compartments of approximately equal volume, one with a large diffusion coefficient (1.70 +/- 0.12 x 10(-5) cm2/s) and a second with a significantly smaller diffusion coefficient (0.25 +/-0.01 x 10(-5) cm2/s). We propose that these two experimentally distinguishable compartments correspond to the extra- and intracellular regions, respectively, of the viable rim of the spheroid. Although the diffusion coefficients were significantly different in the medium, the necrotic center, and the viable rim, the activation energy for diffusion was the same in the three regions (0.20 eV). Studies of perfused spheroids at 37-degrees-C show the same dependence of the diffusion coefficients on the diffusion filter as observed for unperfused spheroids at 22-degrees-C. These results demonstrate the ability of nuclear magnetic resonance microimaging to investigate diffusion at the cellular level, which will lead to a better understanding of microenvironmental regulation in tumors.

Studies of self-diffusion by magnetic resonance imaging using variations of the pulsed-gradient spin-echo experiment are complicated by the presence of the imaging-gradient pulses. This problem is particularly severe in NMR microscopy, where the diffusion gradients are of the same order or even smaller than the imaging gradients. Due to cross terms between the diffusion gradient and parallel imaging gradients, the Stejskal-Tanner relation no longer applies. This commonly used equation could result in significant overestimation of the self-diffusion coefficient when used in such instances. The effect of diffusion on signal attenuation in a number of spin-echo diffusion imaging sequences has been analyzed, and analytical expressions including the cross terms with the imaging gradients have been derived. The equations derived were verified experimentally through measurements of the self-diffusion coefficient of water, using high-resolution microimaging (imaging gradients of 10–15 G/cm, pixel size of 23 μm) at 400 MHz. Including the cross terms in the data analysis yields values within the literature range (2.6 × 10−5 cm2/s) for the self-diffusion coefficient of water. Neglecting the cross terms is demonstrated to result in a tenfold over-estimation of the diffusion coefficient. When imaging gradients which are larger than the incremented diffusion gradient are used, the experiment becomes significantly more sensitive to diffusion effects, due to the cross terms between the gradients. This predicted and observed result improves the accuracy of imaging diffusion experiments and may also be applicable in spectroscopic diffusion measurements.