Placental growth factor deficiency is associated with impaired cerebral vascular development in mice.

STUDY HYPOTHESIS: Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning. STUDY FINDING: PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development. WHAT IS KNOWN ALREADY: PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf  (-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf  (-/-) mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf  (-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf  (-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgf  (-/-) brains. At E14.5, Pgf  (-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgf  (-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not. LIMITATIONS, REASONS FOR CAUTION: Since Pgf  (-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring. WIDER IMPLICATIONS OF THE FINDINGS: These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queen's University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests.

Effects of Sildenafil Citrate and Heparin Treatments on Placental Cell Morphology in a Murine Model of Pregnancy Loss.

Lipopolysaccharide (LPS) injections during pregnancy are well established as models for pregnancy complications, including fetal growth restriction (FGR), thrombophilia, preterm labor and abortion. Indeed, inflammation, as induced by LPS injection has been described as a pivotal factor in cases of miscarriage related to placental tissue damage. The phosphodiesterase-5 inhibitor sildenafil (Viagra®) is currently used to treat FGR cases in women, while low-molecular weight heparin (Fragmin®) is a standard treatment for recurrent miscarriage (RM). However, the pathways and cellular dynamics involved in RM are not completely understood. The aim of this study was to evaluate the protective effect of sildenafil and dalteparin in a mouse model of LPS-induced abortion. Histopathology, ultrastructural analysis and immunofluorescence for P-selectin were studied in two different placental cell types: trophoblast cells and labyrinth endothelial cells. Treatment with sildenafil either alone or in combination with heparin showed the best response against LPS-induced injury during pregnancy. In conclusion, our results support the use of these drugs as future therapeutic agents that may protect the placenta against inflammatory injury in RM events. Analyses of the ultrastructure and placental immunophysiology are important to understand the mechanism underlying RM. These findings may spark future studies and aid in the development of new therapies in cases of RM.

Effect of the combination of metformin hydrochloride and melatonin on oxidative stress before and during pregnancy, and biochemical and histopathological analysis of the livers of rats after treatment for polycystic ovary syndrome.

The aim of the present study was to analyze the effect of a combination of metformin hydrochloride and melatonin on oxidative stress together with a biochemical and histopathological analysis of the livers of Wistar rats induced with PCOS. The results indicated that a combination of the drugs was more effective in the reduction of plasmatic levels of liver enzyme alanine aminotransferase, nitric oxide and total glutathione, and decreased the inflammatory response and histopathological damage, producing results that were significantly similar to animals from the control group. A mixture of the drugs produced more effective results against liver toxicity caused by PCOS, encouraging the normalization of biochemical parameters. During pregnancy, there was reduced oxidative stress compared to monotherapeutic use of these drugs. Interestingly, the combination of the drugs caused a physiological reaction similar to responses identified in healthy rats without induction of the PCOS control group. However, the clinical and physiological effectiveness of the combination should be further explored, especially with respect to the possible side effects on offspring.

Diethylcarbamazine reduces chronic inflammation and fibrosis in carbon tetrachloride- (CCl4-) induced liver injury in mice.

This study investigated the anti-inflammatory effects of DEC on the CCl4-induced hepatotoxicity in C57BL/6 mice. Chronic inflammation was induced by i.p. administration of CCl4 0.5 µL/g of body weight through two injections a week for 6 weeks. DEC (50 mg/kg) was administered by gavage for 12 days before finishing the CCl4 induction. Histological analyses of the DEC-treated group exhibited reduced inflammatory process and prevented liver necrosis and fibrosis. Immunohistochemical and immunofluorescence analyses of the DEC-treated group showed reduced COX-2, IL1ß, MDA, TGF-ß, and αSMA immunopositivity, besides exhibiting decreased IL1ß, COX-2, NFκB, IFNγ, and TGFß expressions in the western blot analysis. The DEC group enhanced significantly the IL-10 expression. The reduction of hepatic injury in the DEC-treated group was confirmed by the COX-2 and iNOS mRNA expression levels. Based on the results of the present study, DEC can be used as a potential anti-inflammatory drug for chronic hepatic inflammation.

Sildenafil (Viagra) protective effects on neuroinflammation: the role of iNOS/NO system in an inflammatory demyelination model.

We recently demonstrated that sildenafil reduces the expression of cytokines, COX-2, and GFAP in a demyelinating model induced in wild-type (WT) mice. Herein, the understandings of the neuroprotective effect of sildenafil and the mediation of iNOS/NO system on inflammatory demyelination induced by cuprizone were investigated. The cerebella of iNOS(-/-) mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil. Cuprizone increased GFAP, Iba-1, TNF- α , COX-2, IL-1 ß , and IFN- γ expression, decreased expression of glutathione S-transferase pi (GSTpi), and damaged myelin in iNOS(-/-) mice. Sildenafil reduced Iba-1, IFN- γ , and IL-1 ß levels but had no effect on the expression of GFAP, TNF- α , and COX-2 compared to the cuprizone group. Sildenafil elevated GSTpi levels and improved the myelin structure/ultrastructure. iNOS(-/-) mice suffered from severe inflammation following treatment with cuprizone, while WT mice had milder inflammation, as found in the previous study. It is possible that inflammatory regulation through iNOS-feedback is absent in iNOS(-/-) mice, making them more susceptible to inflammation. Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition, as its effect on iNOS(-/-) mice was limited. Further studies are required to explain the underlying mechanism of the sildenafil effects.

Sildenafil (Viagra®) down regulates cytokines and prevents demyelination in a cuprizone-induced MS mouse model.

Sildenafil induces cGMP accumulation through phosphodiesterase-5 (PDE5) inhibition. cGMP-pathways protect oligodendrocytes and modulate astroglial and microglial reactions. Microglia and astrocytes play an important role in perpetuating multiple sclerosis (MS), a chronic inflammatory disease characterized by demyelination. Therefore, sildenafil can be a potential tool for MS treatment. The present study investigated the effects of sildenafil on the myelin structure and astrocyte/microglia-mediated neuroinflammation in an animal model of MS. Cuprizone-induced demyelination and neuroinflammation in rodents has been widely used as a model for MS. Herein, five male C57BL/6 mice (7-10 weeks old) were used per group. Over a 4-week period, the different groups received the following: (1) cuprizone (0.2%) mixed into the chow; (2) cuprizone in the chow and sildenafil (Viagra®; 3, 25 or 50mg/kg) in the drinking water; or (3) pure chow and water (control group). Cerebella were analyzed using transmission electron microscopy, western blotting, immunohistochemistry and luxol fast blue staining. Cuprizone induced tissue damage, with an increase in GFAP, Iba-1 and COX-2 and demyelination in comparison to the control group. However, cuprizone did not affect the expression of cytokines (TNF-α, IFN-γ, IL-1ß and IL-2). Sildenafil reduced GFAP (25 and 50mg/kg) and Iba-1 expression (25mg/kg) in comparison to the cuprizone group, indicating the modulation of astrocytes and microglia, respectively. Sildenafil preserved myelin and axons ultrastructure and strongly reduced IFN-γ, TNF-α, IL-1ß, IL-2 and COX-2 expression in comparison to the control and/or cuprizone groups. The results demonstrate a protective effect of sildenafil in the cerebellum. Thus, well-designed clinical trials may demonstrate that the oral administration of sildenafil can be suitable for individuals with MS and other neuroinflammatory/neurodegenerative diseases, providing additional benefits to current treatments.

RNA binding protein, tristetraprolin in a murine model of recurrent pregnancy loss.

Recurrent pregnancy loss is a major reproductive pathology affecting 1-5% of pregnant women worldwide. A distinct feature of this reproductive pathology is involvement of key inflammatory cytokines and transcription factors such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and nuclear factor kappa beta (NF-κB). Special classes of RNA-binding proteins regulate the transcripts of many of these important cytokines and regulatory factors via binding to the 3' untranslated regions (UTRs) and/or poly(A) tail and destabilizing/stabilizing the transcript. The tristetraprolin (TTP/ZFP36) family have been found to be potent destabilizers of the aforementioned inflammatory and cellular response cytokines. The aim of this study was to evaluate whether tristetraprolin is expressed in the placenta and involved in modulating inflammation in mouse model of lipopolysaccharide (LPS)-induced fetal loss. In this study, Swiss-albino mice were injected with LPS at gestational day 15.5 and placental tissues were harvested 6 hours post-LPS injection. Histopathology and immunohistochemistry analyses clearly revealed cellular stress and death in LPS treated placentas compared to controls. TTP protein was downregulated, while targets TNF-α and IL-6 were upregulated in LPS group compared to controls. We observed increased TTP nuclear immunolocalization corresponding with higher NF-κB nuclear localization in trophoblasts from LPS treated placentas. Our results suggest that RNA-binding proteins such as TTP are expressed and perhaps involved in the modulation of inflammation-induced pregnancy pathologies.

Involvement of AMPK, IKßα-NFκB and eNOS in the sildenafil anti-inflammatory mechanism in a demyelination model.

Sildenafil (Viagra®) has recently been found to have a neuroprotective effect, which occurs through the inhibition of inflammation and demyelination in the cerebellum. However, the mechanism of action of sildenafil remains unknown. AMPK, the regulatory protein of the lipid and glucose metabolism, plays a protective role by activating the eNOS enzyme. The production of a nanomolar concentration of NO by eNOS has an anti-inflammatory effect through the cGMP signaling pathway and plays an important role in the regulation of the nuclear transcription factor (NFkB), preventing the expression of inflammatory genes. The present study investigated whether AMPK-eNOS-NO-cGMP-IКßα-NFkB is involved in the mechanism of action of sildenafil in a cuprizone-demyelination model. Neuroinflammation and demyelination induced by cuprizone in rodents have been widely used as a model of MS. In the present study, five male C57BL/6 mice (7-10 weeks old) were used. Over a four week period, the groups received: cuprizone (CPZ) 0.2% mixed in feed; CPZ in the diet, combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently (sild-T0) or 15 days (sild-T15) after the start of CPZ. Control animals received pure food and water. The cerebella of the mice were dissected and processed for immunohistochemistry, immunofluorescence (frozen), western blotting and dosage of cytokines (Elisa). CPZ induced an increase in the expression of GFAP, IL-1ß TNF-α, total NFkB and inactive AMPK, and prompt microglia activation. CPZ also induced a reduction of IKßα. The administration of sildenafil reduced the expression of the pro-inflammatory cytokines IL-1ß and TNF-α and increased the expression of the anti-inflammatory cytokine IL-10. In addition, the administration of sildenafil reduced expression of GFAP, NFkB, inactive AMPK and iNOS, and increased IKßα. Interestingly, sildenafil also reduced levels of NGF. In general, the sild-T0 group was more effective than sild-T15 in improving clinical status and promoting the control of neuroinflammation. The present study offers evidence that sildenafil has anti-inflammatory and neuroprotective effects, which are probably achieved through modulation of AMPK-IKßα-NFκB signaling. In addition, eNOS may play a role in the sildenafil neuroprotective mechanism, contributing to the activation of AMPK. However, other pathways such as MAPK-NFkB and the downstream proteins AMPK (AMPK-SIRT1-NFκB) should also be further investigated. An understanding of these mechanisms of action is critical for the clinical use of sildenafil to control neuroinflammation in neurodegenerative diseases such as MS.

Role of iNOS-NO-cGMP signaling in modulation of inflammatory and myelination processes.

Nitric oxide (NO) is the main activator of the soluble guanylate cyclase (sGC)-guanosine 3'5' cyclic monophosphate (cGMP) pathway. The level of cGMP is regulated by phosphodiesterases (PDEs), which break down cGMP. It has been reported that levels of NO in the central nervous system (CNS) can greatly increase during demyelination and/or neuroinflammation. Controversially, in demyelination models, mice without iNOS may develop more severe cases of disease. Furthermore, cGMP accumulation caused by PDE inhibitors has an anti-inflammatory/neuroprotective effect in MS-models. The role of the NO-cGMP pathway in the nervous tissue is, therefore, complex and not fully understood. The aim of the present study was to contribute to existing knowledge of the role of this pathway in the CNS. Wild type (WT - C57BL/6) and iNOS(-/-) animals were treated with sildenafil (25mg/kg) for 8 weeks. Control animals were not treated. VCAM and ICAM (adhesion proteins), GFAP and Iba-1 (astrocyte and microglia markers, respectively), PKG (cGMP-dependent protein kinase), sGC, eNOS (constitutive endothelial NO sinthase) and GSTpi (a marker of mature oligodendrocytes) were evaluated in the cerebellum using immunohistochemistry or western blotting. Myelin was assessed by luxol fast blue staining and electron transmission microscopy. Treatment with sildenafil reduced ICAM and VCAM levels (anti-inflammatory effect) and increased GFAP and Iba-1 expression (clearance phenotype) in WT animals. The expression of VCAM, ICAM, GFAP, PKG and sGC was lower in iNOS(-/-) mice than in WT control animals. The treatment of iNOS(-/-) animals with sildenafil resulted in an increase of all proteins (pro-inflammatory effect). There was overexpression of eNOS in untreated iNOS(-/-) mice. The myelin structure of iNOS(-/-) animals was damaged in comparison with WT control. Sildenafil increased GSTpi and resulted in an improved myelin structure in iNOS(-/-) mice. In conclusion, NO-cGMP signaling plays a role in the regulation of inflammation and myelination processes. The accumulation of cGMP produced opposite effects in WT and iNOS(-/-) mice. This can be explained by the overexpression of eNOS in iNOS(-/-) mice, unbalancing cGMP signaling, or cGMP has a dual role in inflammation. Drugs that modulate the NO-sGC-cGMP pathway may be clinically beneficial in the treatment of neuroinflammatory/demyelinating disorders, but further studies of the regulation of this pathway are required.