Publications

BACKGROUND. The significant risks posed to mothers and fetuses by COVID-19 in pregnancy have sparked a worldwide debate surrounding the pros and cons of antenatal SARS-CoV-2 inoculation, as we lack sufficient evidence regarding vaccine effectiveness in pregnant women and their offspring. We aimed to provide substantial evidence for the effect of the BNT162b2 mRNA vaccine versus native infection on maternal humoral, as well as transplacentally acquired fetal immune response, potentially providing newborn protection. METHODS. A multicenter study where parturients presenting for delivery were recruited at 8 medical centers across Israel and assigned to 3 study groups: vaccinated (n = 86); PCR-confirmed SARS-CoV-2 infected during pregnancy (n = 65), and unvaccinated noninfected controls (n = 62). Maternal and fetal blood samples were collected from parturients prior to delivery and from the umbilical cord following delivery, respectively. Sera IgG and IgM titers were measured using the Milliplex MAP SARS-CoV-2 Antigen Panel (for S1, S2, RBD, and N). RESULTS. The BNT162b2 mRNA vaccine elicits strong maternal humoral IgG response (anti-S and RBD) that crosses the placenta barrier and approaches maternal titers in the fetus within 15 days following the first dose. Maternal to neonatal antiCOVID-19 antibodies ratio did not differ when comparing sensitization (vaccine vs. infection). IgG transfer ratio at birth was significantly lower for third-trimester as compared with second trimester infection. Lastly, fetal IgM response was detected in 5 neonates, all in the infected group. CONCLUSION. Antenatal BNT162b2 mRNA vaccination induces a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.

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 MRIs 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 placentas 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.

Angiogenesis and lymphangiogenesis are key processes during embryogenesis as well as under physiological and pathological conditions. Vascular endothelial growth factor C (VEGFC), the ligand for both VEGFR2 and VEGFR3, is a central lymphangiogenic regulator that also drives angiogenesis. Here, we report that members of the highly conserved BACH (BTB and CNC homology) family of transcription factors regulate VEGFC expression, through direct binding to its promoter. Accordingly, down-regulation of bach2a hinders blood vessel formation and impairs lymphatic sprouting in a Vegfc-dependent manner during zebrafish embryonic development. In contrast, BACH1 overexpression enhances intratumoral blood vessel density and peritumoral lymphatic vessel diameter in ovarian and lung mouse tumor models. The effects on the vascular compartment correlate spatially and temporally with BACH1 transcriptional regulation of VEGFC expression. Altogether, our results uncover a novel role for the BACH/VEGFC signaling axis in lymphatic formation during embryogenesis and cancer, providing a novel potential target for therapeutic interventions.

OBJECTIVE: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood.APPROACH AND RESULTS: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis.CONCLUSIONS: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.

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.

In the article \u201cPassive or Active Immunization with Myelin Basic Protein Promotes Recovery from Spinal Cord Injury Contusion\u201d by Ehud Hauben, Oleg Butovsky, Uri Nevo, Eti Yoles, Gila Moalem, Eugenia Agranov, Felix Mor, Raya Leibowitz-Amit, Evgenie Pevsner, Solange Akselrod, Michal Neeman, Irun R. Cohen, and Michal Schwartz, which appeared on pages 64216430 of the September 1, 2000 issue, the following error was discovered:In the legend of Figure 4A, a reference is missing. Hauben E, Nevo U, Yoles E, Moalem G, Agranov E, Mor F, Akselrod S, Neeman M, Cohen IR, Schwartz M (2000) Autoimmune T cells as potential neuroprotective therapy for spinal cord injury. Lancet 355:28287, which is cited in the text of the article, should be cited in the legend as well. The sentence in the legend that reads \u201cFor comparison, a similar experiment using five PBS-treated and six rats treated immediately with anti-MBP T cells is shown here\u201d should be replaced by \u201cFor comparison, a similar experiment, taken from Hauben et al. (2000), of six rats treated immediately with anti-MBP is shown.\u201d

The ovary is a dynamic organ that undergoes dramatic remodeling throughout the ovulatory cycle. Maturation of the ovarian follicle, release of the oocyte in the course of ovulation as well as formation and degradation of corpus luteum involve tightly controlled remodeling of the extracellular matrix and vasculature. Ovarian tumors, regardless of their tissue of origin, dynamically interact with the ovarian microenvironment. Their activity in the tissue encompasses recruitment of host stroma and immune cells, attachment of tumor cells to mesothelial layer, degradation of the extracellular matrix and tumor cell migration. High-resolution dynamic imaging of such processes is particularly challenging for internal organs. The implementation of a novel imaging window as reported here enabled longitudinal microscopy of ovarian physiology and orthotopic tumor invasion.

Oxygen (O₂) plays a central role in most living organisms. The concentration of O₂ is important in physiology and pathology. Despite the importance of accurate knowledge of the O₂ levels, there is very limited capability to measure with high spatial resolution its distribution in millimeter-scale live biological samples. Many of the current oximetric methods, such as oxygen microelectrodes and fluorescence lifetime imaging, are compromised by O₂ consumption, sample destruction, invasiveness, and difficulty to calibrate. Here, we present a new method, based on the use of the pulsed electron spin resonance (ESR) microimaging technique to obtain a 3D mapping of oxygen concentration in millimeter-scale biological samples. ESR imaging requires the incorporation of a suitable stable and inert paramagnetic spin probe into the desirable object. In this work, we use microcrystals of a paramagnetic spin probe in a new crystallographic packing form (denoted tg-LiNc-BuO). These paramagnetic species interact with paramagnetic oxygen molecules, causing a spectral line broadening that is linearly proportional to the oxygen concentration. Typical ESR results include 4D spatial-spectral images that give an indication about the oxygen concentration in different regions of the sample. This new oximetry microimaging method addresses all the problems mentioned above. It is noninvasive, sensitive to physiological oxygen levels, and easy to calibrate. Furthermore, in principle, it can be used for repetitive measurements without causing cell damage. The tissue model used in this research is spheroids of Human Colorectal carcinoma cell line (HCT-116) with a typical diameter of ∼600 μm. Most studies of the microenvironmental O₂ conditions inside such viable spheroids carried out in the past used microelectrodes, which require an invasive puncturing of the spheroid and are also not applicable to 3D O₂ imaging. High resolution 3D oxygen maps could make it possible to evaluate the relationship between morphological and physiological alterations in the spheroids, which would help understand the oxygen metabolism in solid tumors and its correlation with the susceptibility of tumors to various oncologic treatments.

Ovulation and inflammation share common attributes, including immune cell invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and fluorescence-activated cell sorting analyses, we demonstrate that CD11c-positive, F4/80-negative cells, representing DCs, are recruited to the ovary under gonadotropin regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of the cumulus-oocyte complex, release of the ovum from the ovarian follicle, formation of a functional corpus luteum, and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DC transplantation after conditional ablation of CD11cpositive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by up-regulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. In addition to discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classic immunologic role, which is relevant also to other biological systems.

Angiogenesis is a hallmark of cancer, and its noninvasive visualization and quantification are key factors for facilitating translational anticancer research. Using four tumor models characterized by different degrees of aggressiveness and angiogenesis, we show that the combination of functional in vivo and anatomical ex vivo X-ray micro-computed tomography (μCT) allows highly accurate quantification of relative blood volume (rBV) and highly detailed three-dimensional analysis of the vascular network in tumors. Depending on the tumor model, rBV values determined using in vivo μCT ranged from 2.6% to 6.0%, and corresponds well with the values assessed using IHC. Using ultra-high-resolution ex vivo μCT, blood vessels as small as 3.4 μm and vessel branches up to the seventh order could be visualized, enabling a highly detailed and quantitative analysis of the three-dimensional micromorphology of tumor vessels. Microvascular parameters such as vessel size and vessel branching correlated very well with tumor aggressiveness and angiogenesis. In rapidly growing and highly angiogenic A431 tumors, the majority of vessels were small and branched only once or twice, whereas in slowly growing A549 tumors, the vessels were much larger and branched four to seven times. Thus, we consider that combining highly accurate functional with highly detailed anatomical μCT is a useful tool for facilitating high-throughput, quantitative, and translational (anti-) angiogenesis and antiangiogenesis research.

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.

Multimodal imaging is an important part of the study of placenta structure and function, as well as embryonic development, particularly in transgenic mice. The development of novel imaging techniques can contribute significantly to enhance our understanding of placental structure, exchange within the placenta, as well as the architecture and function of the maternal and fetal vasculature system, all of which are critical in the evaluation of the dynamic relationships between the mother, placenta, and fetus during pregnancy. Recent progress of a number of imaging approaches is described herein, including non-invasive MRI, ultrasound imaging, fluorescence microscopy, and photoacoustic imaging. Applications of these techniques are used to monitor the details of blood-flow patterns in the uterine arteries, to measure placental perfusion, and to characterize angiogenesis and vascular permeability, providing insight into placental and fetal pathologies. All of these findings eventually provide a better appreciation for the unique properties of mammalian development and reproduction.

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.

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE- 777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.

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 iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow-up of transgenic mice that chronically over-expressed the heavy chain of ferritin (h-ferritin) in liver hepatocytes (liver-hfer mice) over a period of 2 years, with the aim of investigating the long-term effects of elevated level of h-ferritin on MR signal and on the well-being of the mice. Analysis revealed that aging liver-hfer mice, exposed to chronic elevated expression of h-ferritin, have increased R₂ values compared to WT. As expected for ferritin, R₂ difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over-expression, and support the use of h-ferritin as a reporter gene for MRI.

During the early stages of angiogenesis, following stimulation of endothelial cells by vascular endothelial growth factor (VEGF), the vascular wall is breached, allowing high molecular weight proteins to leak from the vessels to the interstitial space. This hallmark of angiogenesis results in deposition of a provisional matrix, elevation of the interstitial pressure and induction of interstitial convection. Albumin, the major plasma protein appears to be an innocent bystander that is significantly affected by these changes, and thus can be used as a biomarker for vascular permeability associated with angiogenesis. Traditionally, albumin leak in superficial organs was followed by colorimetry or morphometry with the use of albumin binding vital dyes. Over the last years, the introduction of tagged-albumin that can be detected by various imaging methods, such as magnetic resonance imaging and positron emission tomography, opened new possibilities for quantitative three dimension dynamic analysis of permeability in any organ. Using these tools it is now possible to follow not only vascular permeability, but also interstitial convection and lymphatic drain. Active uptake of tagged albumin by caveolae-mediated endocytosis opens the possibility for using labeled albumin for vital staining of cells and cell tracking. This approach was used for monitoring recruitment of perivascular stroma fibroblasts associated with tumor angiogenesis.

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.

Keywords: Developmental Biology

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.

Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-β1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.

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.

Uterine receptivity and embryo implantation depend on local induction of angiogenesis and vascular permeability. Poor uterine receptivity has been implicated in implantation failure; however, relatively little is known about the mechanism that underlies endometrial vascular hyperpermeability in implantation sites. Here we show that contrast-enhanced (CE)-MRI and fluorescence microscopy using biotin-BSA-GdDTPA allowed high-resolution detection and quantitative assessment of mouse embryo implantation sites as early as embryonic day 4.5 (E4.5), and subsequent vascular expansion at E5.5. Vessel permeability, but not blood volume, was significantly elevated in E4.5 implantation sites relative to nonimplanted uterus, showing that elevation of vascular permeability is a very early response preceding E4.5. A significantly increased blood volume was detected by MRI and fluorescence microscopy in implantation sites between E4.5 and E5.5. On the other hand, despite the increase in blood volume, implantation sites showed only a small nonsignificant further increase in vascular permeability during these 2 days, demonstrating the rapid dynamics of vascular remodeling during the early days of pregnancy. Functional imaging by MRI, as reported here, allows multiparametric measurement of angiogenesis during normal mouse implantation and would facilitate the application of MRI to evaluate involvement of the vasculature in mouse models of impaired implantation.

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.

Maintaining homogeneous perfusion in tissues undergoing remodeling and vascular expansion requires tight orchestration of the signals leading to endothelial sprouting and subsequent recruitment of perivascular contractile cells and vascular maturation. This regulation, however, is frequently disrupted in tumors. We previously demonstrated the role of tumor-associated myofibroblasts in vascularization and exit from dormancy of human ovarian carcinoma xenografts in nude mice. The aim of this work was to determine the contribution of stroma and tumor cell-derived angiogenic growth factors to the heterogeneity of vascular permeability and maturation in MLS human ovarian carcinoma tumors. We show by RT-PCR and by in situ hybridization that VEGF was expressed by the tumor cells, while angiopoietin-1 and 2 were expressed only by the infiltrating host stroma cells. Vascular maturation was detected in vivo by vasoreactivity to hypercapnia, measured by BOLD contrast MRI and validated by immunostaining of histologic sections to alpha-smooth muscle actin. Vascular permeability was measured in vivo by dynamic contrast-enhanced MRI using albumin-based contrast material and validated in histologic sections by fluorescent staining of the biotinylated contrast material. MRI as well as histologic correlation maps between vascular maturation and vascular permeability revealed a wide range of vascular phenotypes, in which the distribution of vascular maturation and vasoreactivity did not overlap spatially with reduced permeability. The large heterogeneity in the degree of vascular maturation and permeability is consistent with the differential expression pattern of VEGF and angiopoietins during tumor angiogenesis. (c) 2005 Wiley-Liss, Inc.

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.

Ischemic injury and revascularization are frequently associated with hyperpermeability. Although extravasation of plasma proteins may promote tissue recovery through the generation of the provisional matrix that supports angiogenesis, edema may also result in progressive damage to the muscle. The aim of this research was to determine the time course of hyperpermeability associated with the angiogenic response induced by ligation of the femoral artery at the right posterior limb in mice. Hyperpermeability was followed noninvasively by MRI using an in-house-built permanent polyethylene catheter that enabled daily intravenous administration of biotin-BSA-Gd-DTPA. The mice were scanned once prior to ligation and five times during the week post-ligation. The MRI data, along with histopathology, indicated that the early hemodynamic compensation over loss of arterial blood supply occurred by angiogenesis and dilation of vessels in the skin and subcutaneous fat, and was accompanied by vascular hyperpermeability around the site of ligation. Functional recovery of the ischemic limb (i.e., regaining the ability to step on the limb), and the color and shape of the toes correlated with regeneration as shown by histopathology and MRI analysis. Thus, MRI provided valuable information on the transient hyperpermeability induced during the early stages of angiogenesis, and its subsequent resolution along with functional recovery from acute hind limb ischemia in mice.

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) is widely applied for functional imaging of the microcirculation and for functional and structural studies of the microvasculature. The interest in the capabilities of MRI in noninvasively monitoring changes in vascular structure and function expanded over the past years, with specific efforts directed toward the development of novel imaging methods for quantification of angiogenesis. Molecular imaging approaches hold promise for further expansion of the ability to characterize the microvasculature. Exciting applications for MRI are emerging in the study of the biology of microvessels and in the evaluation of potential pharmaceutical modulators of vascular function and development, and preclinical MRI tools can serve for the design of mechanism-of-action-based noninvasive clinical methods for monitoring response to therapy. The aim of this review is to provide a current snapshot of recent developments in this rapidly evolving field.

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.

Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA₂₃ 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability.

Angiogenesis, the growth of new blood vessels, is a critical component in the development of solid tumors. Over the last decade, progress in the study of the biology of angiogenesis has led to identification of a large number of molecules that promote, participate, and regulate the growth of new vessels in normal tissue and in tumors. Consequently, many new targets for suppression of angiogenesis have been identified and are now at various stages of development and evaluation in clinical trials. Magnetic resonance imaging (MRI) provides an attractive tool for in vivo analysis of the basic biology of angiogenesis, for preclinical evaluation of the activity of a number of potential antiangiogenic agents, as well as for clinical detection, diagnosis, and prognosis. One of the features of MRI is the wide range of physiologic parameters by which angiogenesis can be imaged. This review presents the biological basis of angiogenesis with emphasis on characteristics of the neovasculature that can be used for imaging, followed by an overview of the MRI approaches that are being evaluated for the analysis of tumor angiogenesis.

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.

Halofuginone, an inhibitor of collagen α1(l) gene expression was used for the treatment of subcutaneously implanted C6 glioma tumors. Halofuginone had no effect on the growth of C6 glioma spheroids in vitro, and these spheroids showed no collagen α1(l) expression and no collagen synthesis. However, a significant attenuation of tumor growth was observed in vivo, for spheroids implanted in CD-1 nude mice which were treated by oral or intraperitoneal (4 μg every 48 hours) administration of halofuginone. In these mice, treatment was associated with a dose-dependent reduction in collagen α1(l) expression and dose- and time-dependent inhibition of angiogenesis, as measured by MRI. Moreover, halofuginone treatment was associated with improved re-epithelialization of the chronic wounds that are associated with this experimental model. Oral administration of halofuginone was effective also in intervention in tumor growth, and here, too, the treatment was associated with reduced angiogenic activity and vessel regression. These results demonstrate the important role of collagen type I in tumor angiogenesis and tumor growth and implicate its role in chronic wounds. Inhibition of the expression of collagen type I provides an attractive new target for cancer therapy.

The goal of this study was to monitor the vascular bed during the lag phase in growth of implanted spheroids as a model of tumor dormancy. Vascular development and tumor growth were followed up by magnetic resonance imaging in a model system of MLS ovarian carcinoma spheroids implanted subcutaneously in female nude mice. Apparent vessel density in a 1-mm rim surrounding the spheroid was evaluated by gradient echo imaging as a measure of the angiogenic potential of the tumor. Vascular functionality and maturation were assessed by signal intensity changes in response to hyperoxia (elevated oxygen) and hypercapnia (elevated carbon dioxide), respectively. Tumor growth was delayed by 12 to 57 days after implantation. During this long period in which tumor volume did not change, up to 6 cycles of vascular development and regression were observed. We propose here that dynamic remodeling of the vascular bed may precede exit of tumors from dormancy. The sustained oscillations in the angiogenic response to the implanted spheroid are consistent with hypoxic regulation of vascular endothelial growth factor (VEGF), combined with the role of VEGF as an essential survival factor for newly formed blood vessels. Vascular maturation, manifested by physiological vasodilatory response to carbon dioxide, may be important for conferring vascular stability and exit from dormancy.

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

Cyclocreatine (CY), an analogue of creatine, inhibits tumor growth in vivo and proliferation of tumor cells in vitro. The goal of this study was to probe the mechanism of CY transport and cytotoxicity in C6 rat glioma cells and OC238 human ovarian carcinoma cells (creatine kinase activities of 0.16 and 0.016 units/mg protein, respectively). In both cell lines, CY significantly inhibited cell growth with no effect on membrane integrity and on the content of nucleoside triphosphates. An intrinsic ³¹P-nuclear magnetic resonance (³¹P-NMR) signal of phosphocreatine, as well as accumulation of phosphocyclocreatine (PCY) after addition of CY, was observed for C6 glioma but not for the OC238 cells. Transport of CY in C6 glioma showed Michaelis-Menten kinetics for an active sodium-dependent component. Transport was reduced more than fivefold in low-glucose medium. The toxicity of CY to C6 glioma cells may be due to PCY accumulation and cellular swelling. Another mechanism must be invoked to explain CY effects on the human ovarian cancer cells in which no PCY accumulation could be detected and no cellular swelling was observed.

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 1015 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.