Chronic VEGF Blockade Worsens Glomerular Injury in the Remnant Kidney Model

VEGF inhibition can promote renal vascular and parenchymal injury, causing proteinuria, hypertension and thrombotic microangiopathy. The mechanisms underlying these side effects are unclear. We investigated the renal effects of the administration, during 45 days, of sunitinib (Su), a VEGF receptor inhibitor, to rats with 5/6 renal ablation (Nx). Adult male Munich-Wistar rats were distributed among groups S+V, sham-operated rats receiving vehicle only; S+Su, S rats given Su, 4 mg/kg/day; Nx+V, Nx rats receiving V; and Nx+Su, Nx rats receiving Su. Su caused no change in Group S. Seven and 45 days after renal ablation, renal cortical interstitium was expanded, in association with rarefaction of peritubular capillaries. Su did not worsen hypertension, proteinuria or interstitial expansion, nor did it affect capillary rarefaction, suggesting little angiogenic activity in this model. Nx animals exhibited glomerulosclerosis (GS), which was aggravated by Su. This effect could not be explained by podocyte damage, nor could it be ascribed to tuft hypertrophy or hyperplasia. GS may have derived from organization of capillary microthrombi, frequently observed in Group Nx+Su. Treatment with Su did not reduce the fractional glomerular endothelial area, suggesting functional rather than structural cell injury. Chronic VEGF inhibition has little effect on normal rats, but can affect glomerular endothelium when renal damage is already present.

An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats

Previous studies revealed the presence of LTC4 synthase in paraventricular vasopressinergic neurons, suggesting a role for leukotrienes (LTs) in certain neuroendocrine system functions. Our aim was to study the effect of an inhibitor of LT synthesis in the release of arginine vasopressin (AVP) following an osmotic stimulus in rats. Male Wistar rats received an intra-cerebroventricular injection of 2 mu l of the LT synthesis inhibitor MK-886 (1, 2, or 4 mu g/kg), or vehicle (DMSO 5%), 1 h before an intraperitoneal injection of hypertonic saline (NaCl 2 M) or isotonic saline (NaCl 0.01 M) in a volume corresponding to 1% of body weight. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for determining hematocrit, plasma osmolality and plasma AVP levels. As expected, the injection of hypertonic saline significantly increased (P<0.05) the hematocrit, plasma osmolality and plasma AVP levels. While inhibiting LT synthesis by central administration of MK-886 did not cause any additional increase in hematocrit or osmolality, plasma AVP levels were augmented (P<0.05). We conclude that central leukotrienes may have a modulatory role in AVP secretion following an osmotic stimulus, this deserving future studies. (C) 2012 Elsevier B.V. All rights reserved.

Divergent Roles of the CRH Receptors in the Control of Gonadotropin Secretion Induced by Acute Restraint Stress at Proestrus

CRH has been implicated as a mediator of stress-induced effects on the hypothalamus-pituitary-gonad axis, acting via CRH receptors in various brain regions. We investigated whether the effects of restraint stress on the secretion of gonadotropins on the morning of proestrus are mediated by the CRH-R1 or CRH-R2 receptors in the oval subdivision of the anterolateral BST, the central amygdala, the locus coeruleus (LC), or the A1 and A2 neuron groups in the medulla. At proestrus morning, rats were injected with antalarmin (a CRH-R1 antagonist), asstressin2-B (a CRH-R2 antagonist) or vehicles. Thirty minutes after the injection, the animals were placed into restraints for 30 min, and blood was sampled for 2 h. At the end of the experiment, the brains were removed for immunofluorescence analyses. Restraint stress increased the levels of FSH and LH. Antalarmin blocked the stress-induced increases in FSH and LH secretion, but astressin2-B only blocked the increase in FSH secretion. LC showed intense stress-induced neuronal activity. FOS/tyrosine-hydroxylase coexpression in LC was reduced by antalarmin, but not astressin2-B. The CRH-R1 receptor, more than CRH-R2 receptor, appears to be essential for the stimulation of the hypothalamus-pituitary-gonad axis by acute stress; this response is likely mediated in part by noradrenergic neurons in the LC. We postulate that the stress-induced facilitation of reproductive function is mediated, at least in part, by CRH action through CRH-R1 on noradrenaline neurons residing in the LC that trigger GnRH discharge and gonadotropin secretion. (Endocrinology 153: 4838-4848, 2012)

Allelic variations in the vitamin D receptor gene, insulin secretion and parents' heights are independently associated with height in obese children and adolescents

Polymorphisms in the VDR gene were reported to be associated with variations in intrauterine and postnatal growth and with adult height, but also with other traits that are strongly correlated such as the BMI, insulin sensitivity, insulin secretion and hyperglycemia. Here, we assessed the impact of VDR polymorphisms on body height and its interactions with obesity- and glucose tolerance-related traits in obese children and adolescents. We studied 173 prepubertal (Tanner's stage 1) and 146 pubertal (Tanner's stages 2-5) obese children who were referred for a weight-loss program. Three single nucleotide polymorphisms were genotyped: rs1544410 (BsmI), rs7975232 (ApaI) and rs731236 (TaqI). BsmI and TaqI genotypes were significantly associated with height in pubertal children, but the associations did not reach statistical significance in prepubertal children. In stepwise regression analyses, the lean body mass, insulin secretion, BsmI or TaqI genotypes and the father's and the mother's height were independently and positively associated with height in pubertal children. These covariables accounted for 46% of the trait variance. The height of homozygous carriers of the minor allele of BsmI was 0.65 z-scores (4 cm) higher than the height of homozygous carriers of the major allele (P=.0006). Haplotype analyses confirmed the associations of the minor alleles of BsmI and TaqI with increased height. In conclusion, VDR genotypes were significantly associated with height in pubertal obese children. The associations were independent from the effects of confounding traits, such as the body fat mass, insulin secretion, insulin sensitivity and glucose tolerance. (C) 2012 Elsevier Inc. All rights reserved.

HIV-1 induces NALP3-inflammasome expression and interleukin-1 beta secretion in dendritic cells from healthy individuals but not from HIV-positive patients

Objective: NALP3-inflammasome is an innate mechanism, alternative to type-1 interferon, which is able to recognize nucleic acids and viruses in the cytoplasm and to induce pro-inflammatory response. Here, we hypothesized the involvement of inflammasome in the early defense against HIV-1 and in the full maturation of dendritic cells: for this, we evaluated the response of dendritic cells pulsed with HIV-1 in terms of inflammasome activation in healthy donors. Moreover, inflammasome response to HIV was evaluated in HIV-infected individuals. Design and methods: Monocyte-derived dendritic cells isolated from 20 healthy individuals (HC-DC) and 20 HIV-1-infected patients (HIV-DC) were pulsed with alditrithiol-2-inactivated HIV-1. We then analyzed inflammasome genes expression and interleukin-1 beta (IL-1 beta) secretion. Results: In HC-DC, HIV-1 induced higher NLRP3/NALP3 mRNA expression compared with other inflammasome genes such as NALP1/NLRP1 or IPAF/NLRC4 (P < 0.001). This augmented expression was accompanied by CASP1-increased and IL1B-increased mRNA levels and by a significant increment of IL-1b secretion (P < 0.05). Otherwise, HIV-1 failed to activate inflammasome and cytokine production in HIV-DC. HIV-DC showed an increased NLRP3/NALP3 basal expression, suggesting a chronic inflammatory profile of patients' immune cells. Conclusion: HIV-1 was able to induce a NALP3-inflammasome response in healthy individuals, indicating that this inflammasome could play a role in the first steps of HIV-1 infection; the consequent inflammatory process may be important for directing host immune response against the virus and/or disease progression. HIV-DC seemed to be chronically activated, but unresponsive against pathogens. Our findings could be of interest considering the ongoing research about dendritic cell manipulation and therapeutic strategies for AIDS involving dendritic cell-based immune-vaccines. (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Direct effect of PGF2 alpha pulses on PRL pulses, based on inhibition of PRL or PGF2 alpha secretion in heifers

The relationships between PRL and PGF(2 alpha) and their effect on luteolysis were studied. Heifers were treated with a dopamine-receptor agonist (bromocriptine; Bc) and a Cox-1 and -2 inhibitor (flunixin meglumine [FM]) to inhibit PRL and PGF(2 alpha), respectively. The Bc was given (Hour 0) when ongoing luteolysis was indicated by a 12.5% reduction in CL area (cm(2)) from the area on Day 14 postovulation, and FM was given at Hours 0, 4, and 8. Blood samples were collected every 8-h beginning on Day 14 until Hour 48 and hourly for Hours 0 to 12. Three groups of heifers in ongoing luteolysis were used: control (n = 7), Bc (n = 7), and FM (n = 4). Treatment with Bc decreased (P < 0.003) the PRL concentrations averaged over Hours 1 to 12. During the greatest decrease in PRL (Hours 2-6), LH concentrations were increased. Progesterone concentrations averaged over hours were greater (P < 0.05) in the Bc group than in the controls. In the FM group, no PGFM pulses were detected, and PRL concentrations were reduced. Concentrations of PGFM were not reduced in the Bc group, despite the reduction in PRL. Results supported the hypothesis that a decrease (12.5%) in CL area (cm(2)) is more efficient in targeting ongoing luteolysis (63%) than using any day from Days 14 to >= 19 (efficiency/day, 10-24%). The hypothesis that PRL has a role in luteolysis was supported but was confounded by the known positive effect of LH on progesterone. The hypothesis was supported that the synchrony of PGFM and PRL pulses represents a positive effect of PGF(2 alpha), on PRL, rather than an effect of PRL on PGF(2 alpha). (C) 2012 Elsevier Inc. All rights reserved.

The Regulation of Rasd1 Expression by Glucocorticoids and Prolactin Controls Peripartum Maternal Insulin Secretion

The transition from gestation to lactation is characterized by a robust adaptation of maternal pancreatic beta-cells. Consistent with the loss of beta-cell mass, glucose-induced insulin secretion is down-regulated in the islets of early lactating dams. Extensive experimental evidence has demonstrated that the surge of prolactin is responsible for the morphofunctional remodeling of the maternal endocrine pancreas during pregnancy, but the precise molecular mechanisms by which this phenotype is rapidly reversed after delivery are not completely understood. This study investigated whether glucocorticoid-regulated expression of Rasd1/Dexras, a small inhibitoryGprotein, is involved in this physiological plasticity. Immunofluorescent staining demonstrated that Rasd1 is localized within pancreatic beta-cells. Rasd1 expression in insulin-secreting cells was increased by dexamethasone and decreased by prolactin. In vivo data confirmed that Rasd1 expression is decreased in islets from pregnant rats and increased in islets from lactating mothers. Knockdown of Rasd1 abolished the inhibitory effects of dexamethasone on insulin secretion and the protein kinase A, protein kinase C, and ERK1/2 pathways. Chromatin immunoprecipitation experiments revealed that glucocorticoid receptor (GR) and signal transducer and activator of transcription 5b (STAT5b) cooperatively mediate glucocorticoid-induced Rasd1 expression in islets. Prolactin inhibited the stimulatory effect of GR/STAT5b complex on Rasd1 transcription. Overall, our data indicate that the stimulation of Rasd1 expression by glucocorticoid at the end of pregnancy reverses the increased insulin secretion that occurs during pregnancy. Prolactin negatively regulates this pathway by inhibiting GR/STAT5b transcriptional activity on the Rasd1 gene. (Endocrinology 153: 3668-3678, 2012)

Maternal Moderate Physical Training during Pregnancy Attenuates the Effects of a Low-Protein Diet on the Impaired Secretion of Insulin in Rats: Potential Role for Compensation of Insulin Resistance and Preventing Gestational Diabetes Mellitus

The effects of pregestational and gestational low-to-moderate physical training on insulin secretion in undernourished mothers were evaluated. Virgin female Wistar rats were divided into four groups as follows: control (C, n = 5); trained (T, n = 5); low-protein diet (LP, n = 5); trained with a low-protein diet (T + LP, n = 5). Trained rats ran on a treadmill over a period of 4 weeks before mate (5 days week(-1) and 60 min day(-1), at 65% of VO2max). At pregnancy, the intensity and duration of the exercise were reduced. Low-protein groups were provided with an 8% casein diet, and controls were provided with a 17% casein diet. At third day after delivery, mothers and pups were killed and islets were isolated by collagenase digestion of pancreas and incubated for a further 1 h with medium containing 5.6 or 16.7 mM glucose. T mothers showed increased insulin secretion by isolated islets incubated with 16.7 mM glucose, whereas LP group showed reduced secretion of insulin by isolated islets when compared with both C and LP + T groups. Physical training before and during pregnancy attenuated the effects of a low-protein diet on the secretion of insulin, suggesting a potential role for compensation of insulin resistance and preventing gestational diabetes mellitus.

Cediranib: a VEGF receptor tyrosine kinase inhibitor

Cediranib is a potent inhibitor of the VEGF family receptor tyrosine kinases, and a new agent in cancer treatment. The drug has shown promising activity in a variety of solid malignancies, in preclinical models and in clinical trials. Its pharmacokinetics allow for a convenient once-daily administration, with a toxicity profile that is very similar to other VEGF inhibitors. Its main side effects include hypertension, nausea, dysphonia, fatigue and diarrhea. Adverse events seem to be manageable, especially when used in doses lower than 45 mg/day. Studies have shown some activity as a single agent or in combination in advanced tumors, but not enough to secure its approval for routine use up to now. Clinical trials are still evaluating the role of cediranib in combination chemotherapy with cytotoxic agents.

Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes

1. The present study provides the first in vivo evidence that the cannabinoid CB1 receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB1 receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. 2. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB1 receptor. 3. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB1 receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. 4. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB1 receptor in the control of peripheral factors that modulate cardiovascular function. 5. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB1 receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamicpituitaryadrenal axis. 6. Collectively, the results of the present study indicate that the CB1 receptor modulates neurohypophyseal hormone secretion and systemic factors, such as corticosterone and ANP, thus participating in homeostatic responses to altered extracellular volume and plasma tonicity.

Monoclonal antibodies anti-TGF beta 1 and anti-VEGF inhibit the experimental pleurodesis induced by silver nitrate

Background. The mechanisms underlying pleural inflammation and pleurodesis are poorly understood. We hypothesized that the cytokines transforming growth factor beta (TGF beta 1) and vascular endothelial growth factor (VEGF) play a major role in pleurodesis after intrapleural silver nitrate (SN) injection. Method. Forty rabbits received intrapleurally 0.5% SN alone or 0.5% SN + anti-TGF beta 1, anti-IL-8, or anti-VEGF. After 28 days, the animals were euthanized and macroscopic pleural adhesions, microscopic pleural fibrosis, and collagen deposition were analyzed for characterization of the degree of pleurodesis (scores 0-4). Results. Scores of pleural adhesions, pleural fibrosis, total collagen, and thin collagen fibers deposition after 28 days were significantly lower for 0.5% SN + anti-TGF beta 1 and 0.5% SN + anti-VEGF. Significant correlations were found between macroscopic adhesion and microscopic pleural fibrosis with total collagen and thin collagen fibers. Conclusions. We conclude that both TGF beta 1 and VEGF, but not IL-8, mediate the pleural inflammatory response and pleurodesis induced by SN.

In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Correlation between mandibular gland secretion and cuticular hydrocarbons in the stingless bee Melipona quadrifasciata

We investigated whether Melipona quadrifasciata worker mandibular gland secretions contribute directly to their cuticular hydrocarbon profile. The mandibular gland secretion composition and cuticular surface compounds of newly emerged worker bees, nurse bees, and foragers were determined by gas chromatography and mass spectrometry and compared. Both the mandibular gland secretions and the cuticular surface compounds of all worker stages were found to be composed almost exclusively of hydrocarbons. Although the relative proportion of hydrocarbons from the cuticular surface and gland secretion was statistically different, there was a high similarity in the qualitative composition between these structures in all groups of bees.

Actions of the Kunitz-type serine protease inhibitor Amblyomin-X on VEGF-A-induced angiogenesis

Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 mu l; each 48 h) inhibited VEGF-A-induced (10 ng/10 mu l; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-I), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules. (C) 2012 Elsevier Ltd. All rights reserved.

In vivo hydroquinone exposure causes tracheal hyperresponsiveness due to TNF secretion by epithelial cells

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1 h/day for 5 days) and tracheal rings were collected 1 h after the last exposure. HQ exposure caused tracheal hyper-responsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways. (C) 2012 Elsevier Ireland Ltd. All rights reserved.