A FABP4-PPARγ signaling axis regulates human monocyte responses to electrophilic fatty acid nitroalkenes.

Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ signaling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 upregulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upregulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator.

Effects of dexamethasone on estrogen- and pregnancy-induced plasticity in rat uterine sympathetic nerves.

Estrogen and glucocorticoids are known to evoke opposing effects on the uterus. We analyzed the effects of dexamethasone (DEX) on uterine sympathetic denervation elicited by short- and long-term exposure to estrogen of intact prepubertal rats. We also studied the effects of DEX on the physiological degeneration of uterine sympathetic nerves at term pregnancy. Changes in innervation were assessed quantitatively by using computer-assisted methods on uterine cryostat tissue sections stained for tyrosine hydroxylase. At 24 h following treatment of prepubertal rats (25 days of age) with 1 microg or 2.5 microg estrogen, marked increases in uterine size and reductions in the percentage nerve area were observed. Co-administration of DEX (4 mg/kg) attenuated both these short-term estrogen-induced effects. Treatment of 19-day-old rats with a single dose of 25 mug estrogen provoked, at 26 days of age, a 54% reduction in the total nerve area. This reduction was abolished by the co-administration of nine doses of DEX (0.5 mg/kg) at 18-26 days of age. Treatment of rats with the same regime of DEX alone increased the total nerve area by 46% of the control values. Studies of control pregnant rats revealed the unexpected presence of intrauterine nerve fibers at term. Treatment of pregnant rats with six doses of DEX (4 mg/kg) at 16-21 days of age had no effects on the density of uterine sympathetic nerves. These results suggest that DEX has growth-promoting effects on immature uterine sympathetic nerves and may antagonize the degenerative effects elicited by long-term exposure to estrogen.

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA.

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.

The oestrogenized rat myometrium inhibits organotypic sympathetic reinnervation.

Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

Differential effects of oestrogen on developing and mature uterine sympathetic nerves.

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.

Effects of chronic oestrogen treatment are not selective for uterine noradrenaline-containing sympathetic nerves: a transplantation study.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.

The role of NGF in pregnancy-induced degeneration and regeneration of sympathetic nerves in the guinea pig uterus.

In the guinea pig, pregnancy is associated with a generalised depletion of noradrenaline in uterine sympathetic nerves and, in the areas of the uterus surrounding the foetus, by a complete degeneration of sympathetic nerve fibres. These pregnancy-induced changes have been interpreted as a selective effect of placental hormones on the system of short sympathetic fibres arising from the paracervical ganglia. An alternative explanation is that pregnancy affects the neurotrophic capacity of the uterus. We measured NGF-protein levels in the guinea pig uterine horn, tubal end and cervix at early pregnancy, late pregnancy and early postpartum, using a two-site enzyme-linked immunosorbent assay. For comparative purposes the distribution and relative density of noradrenaline-containing sympathetic nerve fibres were assessed histochemically, and tissue levels of noradrenaline were measured biochemically, using high-performance liquid chromatography with electrochemical detection. In all the uterine regions analysed, NGF-protein levels showed a decline at term pregnancy, but in no case was this change statistically significant. After delivery, NGF-protein levels showed a marked increase in the cervix as well as in both the fertile and empty horns. These results suggest that alterations in NGF-protein do not account for the impairment of uterine sympathetic innervation during pregnancy, but may contribute to their recovery after delivery.