Nava Nevo

Cu/Zn Superoxide Dismutase Plays Important Role in Immune Response

Activation of macrophages leads to the secretion of cytokines and enzymes that shape the inflammatory response and increase metabolic processes. This, in turn, results in increased production of reactive oxygen species. The role of Cu/Zn superoxide dismutase (SOD-1), an important enzyme in cellular oxygen metabolism, was examined in activated peritoneal elicited macrophages (PEM) and in several inflammatory processes in vivo. LPS and TNF-α induced SOD-1 in PEM. SOD-1 induction by LPS was mainly via extracellular signal-regulated kinase-1 activation. Transgenic mice overexpressing SOD-1 demonstrated a significant increase in the release of TNF-α and of the metalloproteinases MMP-2 and MMP-9 from PEM. Disulfiram (DSF), an inhibitor of SOD-1, strongly inhibited the release of TNF-α, vascular endothelial growth factor, and MMP-2 and MMP-9 from cultured activated PEM. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. In vivo, transgenic mice overexpressing SOD-1 demonstrated a 4-fold increase in serum TNF-α levels and 2-fold stronger delayed-type hypersensitivity reaction as compared with control nontransgenic mice. Conversely, oral administration of DSF lowered TNF-α serum level by 4-fold, lowered the delayed-type hypersensitivity response in a dose-dependent manner, and significantly inhibited adjuvant arthritis in Lewis rats. The data suggest an important role for SOD-1 in inflammation, establish DSF as a potential inhibitor of inflammation, and raise the possibility that regulation of SOD-1 activity may be important in the treatment of immune-dependent pathologies.

Reactive oxygen species are indispensable in ovulation

Ovulation is stimulated by the preovulatory surge of the pituitary luteinizing hormone (LH). Because the ovulatory response is commonly identified with inflammation, we explored the involvement of reactive oxygen species (ROS) in this process. Our experiments show that administration of broad-range scavengers of oxidative species into the ovarian bursa of mice, hormonally induced to ovulate, significantly reduced the rate of ovulation. LH-induced cumulus mucification/expansion, a necessary requirement for ovulation, was prevented by antioxidants both in vivo and in an ex vivo system of isolated intact ovarian follicles. Along this line, H2O2 fully mimicked the effect of LH, bringing about an extensive mucification/expansion of the follicle-enclosed cumulus–oocyte complexes. Impaired progesterone production was observed in isolated follicles incubated with LH in the presence of the antioxidant agents. Furthermore, LH-stimulated up-regulation of genes, the expression of which is crucial for ovulation, was substantially attenuated upon ROS ablation. This system was also used for demonstrating the role of ROS in phosphorylation and activation of the EGF receptor as well as its downstream effector, p42/44 MAPK. Together, our results provide evidence that ovarian production of ROS is an essential preovulatory signaling event, most probably transiently triggered by LH.

Cu/Zn superoxide dismutase plays a role in angiogenesis.

Endothelial cells produce oxygen radicals spontaneously and this process is augmented by hypoxia/reoxygenation. Cu/Zn superoxide dismutase (SOD‐1) is an important enzyme in cellular oxygen metabolism. To determine whether alterations in SOD‐1 activity affect angiogenesis we used transgenic SOD‐1 (Tg‐SOD) mice with elevated level of SOD‐1. Angiogenesis induced subcutaneously by bFGF in Tg‐SOD mice was 3‐fold higher than in control non‐transgenic (ntg) mice. Oral administration of disulfiram (DSF), an inhibitor of SOD‐1, inhibited angiogenesis in Tg‐SOD mice as well as in CD1 nude mice. Effects of DSF on cultured cells were also tested. Application of DSF to cultured bovine capillary endothelial (BCE) cells caused inhibition of DNA synthesis and induction of apoptosis. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. DSF also reduced the level of glutathione and the production of H2O2 in BCE cells. Moreover, PC12‐SOD cells with elevated SOD‐1 were less sensitive to DSF treatment then control cells. These data indicate that the effects of DSF are mediated by inhibition of SOD‐1 activity. Tumor development is known to largely depend on angiogenesis. We found that oral administration of DSF to mice caused significant inhibition of C6 glioma tumor development and marked reduction (by 10–19‐fold) in metastatic growth of Lewis lung carcinoma. The data suggest a role for SOD‐1 in angiogenesis, establish DSF as a potential inhibitor of angiogenesis and raise the possibility that attenuating SOD‐1 activity may be important in treatment of angiogenesis‐dependent pathologies.

Vascular Remodeling and Angiogenesis in Ectopic Ovarian Transplants: A Crucial Role of Pericytes and Vascular Smooth Muscle Cells in Maintenance of Ovarian Grafts

Abstract 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.

Inhibition of Rat Oocyte Maturation and Ovulation by Nitric Oxide: Mechanism of Action

Established gap junctional communication (GJC) in the ovarian follicle is essential for maintaining the oocytes in meiotic arrest. Alternatively, LH-induced reinitiation of meiosis is subsequent to breakdown of GJC. It was recently reported that nitric oxide (NO) inhibits maturation in rat follicle-enclosed oocytes and elevates GJC in cultured mesangial cells. Taking these observations into account, we hypothesized that NO prevents reinitiation of meiosis by antagonizing the effect of LH on GJC in the ovarian follicle. Indeed, we found that NO interferes with LH-induced disruption of GJC as well as with the decrease of the expression of the gap junction protein GJA1 (previously known as CONNEXIN43). We also demonstrated that NO prevents activation of LH-induced mitogen-activated protein kinases (MAPKs) 1 and 2 and inhibits cumulus expansion. Along this line, incubation of ovarian follicles with an inhibitor of soluble guanylate cyclase, which is a downstream NO effector, induced on its own oocyte maturation as well as cumulus expansion. Unlike previous studies, we show here that elevation of NO resulted in inhibition of ovulation. We conclude that the mechanism by which NO inhibits LH-induced oocyte maturation possibly involves a negative effect on MAPK activation and, in turn, interference with interruption of GJC. This action of NO in the ovarian follicle is apparently mediated by cGMP. In addition, the negative effect of NO on ovulation may be subsequent to its inhibitory effect on cumulus expansion. Together, this study suggests that the preovulatory decrease in NO concentrations is a prerequisite for the ovarian response to LH.

Angiogenesis in ectopic ovarian xenotransplantation: Multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI

It has been suggested that ovarian cryopreservation and xenotransplantation can be used to preserve oocytes from damage during anticancer treatments. The main obstacle to subsequent ovarian grafting is loss of oocytes due to impaired perfusion. The aim of this study was to characterize angiogenic events following ovary xenotransplantation. Rat ovaries were transplanted into or onto the muscle of immunocompromised CD1‐nude mice. Ovariectomy (OVX) of host mice prior to transplantation supported the resumption of follicular development, as manifested by the prevalence of antral follicles and corpora lutea. Two days after transplantation, the grafts were devoid of blood supply. Functional vessels within the graft were detected by MRI and histology from day 7 and on. By 2–3 weeks, both blood volume fraction and permeability in the graft, as measured with the use of albumin‐based MR contrast material, were significantly elevated relative to the adjacent muscle. Extravasation of contrast material from the graft neovasculature was followed by interstitial convection in the muscle surrounding the graft, and draining toward the proximal popliteal lymph node. Development of the vasculature was monitored noninvasively, providing a time scale for revascularization and recovery of ovarian function following xenotransplantation of ovarian grafts. Magn Reson Med 52:741–750, 2004.

Modulation of the pharmacokinetics of macromolecular contrast material by avidin chase: MRI, optical, and inductively coupled plasma mass spectrometry tracking of triply labeled albumin.

The goal of this work was to develop an MRI method for mapping the clearance of interstitial macromolecular plasma proteins after their extravasation from permeable blood vessels. To that end, a well‐defined window of exposure to elevated blood levels was generated by inducing rapid clearance of macromolecular contrast material from the blood. Experimental removal of the intravascular component allowed subsequent tracking of clearance from the interstitial compartment in the absence of further contrast extravasation. The contrast material was based on albumin triply labeled with biotin, fluorescent tag, and GdDTPA, allowing optical, inductively coupled plasma mass spectrometry (ICP‐MS) and MRI detection. The biotin tag was used here for in vivo chasing of the contrast material from the blood by intravenous administration of avidin. Upon administration of avidin the contrast material disappeared from the blood vessels and was cleared by the liver and spleen as detected by MRI, fluorescence of blood samples and histological sections, and by ICP‐MS. Nonbiotinylated fluorescent albumin was not affected by administration of avidin. Contrast material that extravasated from leaky blood vessels in a VEGF overexpressing tumor, prior to administration of avidin, was not cleared by the addition of avidin and showed continued interstitial convection. Thus, avidin‐chase provides an effective tool for in vivo manipulation of the arterial input function by providing experimental control over the rate of clearance of the contrast material from the circulation. Magn Reson Med 50:904–914, 2003.

Cell Lineage Analysis of the Mammalian Female Germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

Glyburide crosses the placenta in vivo in pregnant rats

SummaryNon-insulin-dependent diabetes mellitus (NIDDM) is normally treated by oral hypoglycaemic agents, but their use is excluded during pregnancy because of their potential teratogenic and hypoglycaemic effects on the fetus. This caveat was recently questioned as glyburide was shown to cross an isolated cotyledon in vitro in insignificant amounts. In the present study, placental transport of glyburide in vivo was examined as an indispensable step towards clinical trials. Tritiated glyburide, C14 albumin or C14-labelled diazepam were injected into 13, 9 and 11 pregnant rats, respectively and the radioactivity was measured thereafter in maternal blood and in whole fetal extracts. The ratios between radioactivity in fetal tissue to that in maternal blood for glyburide (0.535 ± 0.068) were similar to those of diazepam (0.641 ± 0.057) which readily crosses the placenta. However, they differed significantly from those for albumin (0.048 ± 0.0004) which does not cross. Moreover, glyburide in fetal tissue consistently reflected its concentration in maternal blood when measured at consecutive intervals after intravenous injection in the mother. In contrast, albumin in fetal tissue was low at all time points regardless of its levels in maternal blood when measured at different times after injection. These data suggest that glyburide crosses the placenta of pregnant rats and should therefore be considered with caution as a hypoglycaemic agent in the treatment of gestational diabetes.

Angiotensin 1-7 as Means to Prevent the Metabolic Syndrome: Lessons From the Fructose-Fed Rat Model

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.