Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide’s protective effects. Moreover, AnxA1−/− mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.

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Phα1β is a potent toxin obtained from the spider Phoneutria nigriventer that blocks neuronal voltage-sensitive Ca(2+) channels. This study compared the antiallodynic effects of Phα1β, ω-conotoxin MVIIA and morphine in mice and their side effects in rats. Mechanical allodynia was measured in mice receiving single intrathecal administration of Phα1β, ω-conotoxin MVIIA or morphine before or after the incisional plantar procedure. The effect of the treatments on cardiovascular profile and global neurological were evaluated in rats. The expression of pro or anti-inflammatory cytokines of human polymorph mononuclear cells was also evaluated. Preemptive use of ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site) induced shorter antiallodynic effect than Phα1β (100 pmol/site) in mice. Post-incision administration of Phα1β (200 pmol/site) induced longer mechanical antiallodynic effect than ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site). Intrathecal injection of Phα1β (200 pmol/site) and morphine (433 pmol/site) did not change while ω-conotoxin MVIIA (100 pmol/site) increased the heart rate in rats 3 h after its administration. Phα1β (200 pmol/site), ω-conotoxin MVIIA (100 pmol/site) and morphine (433 pmol/site) did not change mean arterial pressure 0.5 and 3 h after their administration. The treatments did not alter neurological performance assessed by global neurological evaluation and open-field test. The tested drugs did not induced expression of pro or anti-inflammatory cytokines in CD4 monocytes. In conclusion, preemptive administration Phα1β in mice induced longer antiallodynic effect than ω-conotoxin MVIIA and morphine. Phα1β also induced a longer mechanical antiallodynic effect than ω-conotoxin MVIIA and morphine when used after the surgical incision. The present results suggest that Phα1β has a potential application in the management of postoperative pain with low side effects.

Gold nanoparticle (AuNP) bioconjugates have been used as therapeutic and diagnostic tools; however, in vivo biocompatibility and cytotoxicity continue to be two fundamental issues. The effect of AuNPs (20 nm) conjugated with antibody [immunoglobulin G (IgG)], albumin, protein A, PEG4000, and citrate (cit) were evaluated in vitro using primary human cells of the vascular system. AuNP bioconjugates did not cause lysis of human erythrocytes, apoptosis or necrosis of human leukocytes, and endothelial cells in vitro, although AuNPs had been internalized and detected in the cytoplasm. Moreover, the influence of AuNPs on rheological parameters, blood and vessel wall characteristics was investigated in vivo by intravital microscopy assay using male Wistar rats mesentery microcirculation as model. Intravenous injection of AuNP-IgG or cit-AuNP did not cause hemorrhage, hemolysis or thrombus formation, instead suppressed the leukocyte adhesion to postcapillary vessel walls, an early stage of an inflammatory process. Furthermore, AuNP-IgG abrogated the expression of platelet-endothelial cell adhesion molecule-1, chemotaxis, and oxidative burst activation on neutrophils after leukotriene B4 stimulation, a membrane receptor-dependent stimulus, thus confirming their anti-inflammatory effects in vitro. The expression of oxidative burst activation was also suppressed after stimulating AuNP-IgG-treated neutrophils with lipid-soluble phorbol myristate acetate (PMA), confirming the direct intracellular action of AuNP-IgG on the inflammatory process in vitro. Our in vitro and in vivo experimental approaches highlighted the great potentiality of AuNP bioconjugates for therapeutic and diagnostic applications by parenteral routes.

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 μl; each 48 h) inhibited VEGF-A-induced (10 ng/10 μl; 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-1), 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.

It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

Skin graft successful depends on reduction of local inflammation evoked by the surgical lesion and efficient neovascularization to nutrition the graft. It has been shown that N-terminal portion of the Annexin A1 protein (AnxA1) with its anti-inflammatory properties induces epithelial mucosa repair and presents potential therapeutic approaches. The role of AnxA1 on wound healing has not been explored and we investigated in this study the effect of the peptide Ac2–26 (N-terminal AnxA1 peptide Ac2–26; AnxA12–26) on heterologous skin scaffolds transplantation in BALB/c mice, focusing on inflammation and angiogenesis. Treatment with AnxA12–26, once a day, from day 3–60 after scaffold implantation improved the take of the implant, induced vessels formation, enhanced gene and protein levels of the vascular growth factor-A (VEGF-A) and fibroblast influx into allograft tissue. It also decreased pro- while increasing anti-inflammatory cytokines. The pro-angiogenic activity of AnxA12–26 was corroborated by topical application of AnxA12–26 on the subcutaneous tissue of mice. Moreover, treatment of human umbilical endothelial cells (HUVECs) with AnxA12–26 improved proliferation, shortened cycle, increased migration and actin polymerization similarly to those evoked by VEGF-A. The peptide treatment instead only potentiated the tube formation induced by VEGF-A. Collectively, our data showed that AnxA12–26 treatment favors the tissue regeneration after skin grafting by avoiding exacerbated inflammation and improving the angiogenesis process.

Polyamines (putrescine, spermidine and spermine) are important endogenous regulators of ion channels, such as vanilloid (TRPV1), glutamatergic (NMDA or AMPA/kainate) and acid-sensitive (ASIC) receptors. In the present study, we have investigated the possible nociceptive effect induced by polyamines and the mechanisms involved in this nociception in vivo. The subcutaneous (s.c.) injection of capsaicin (as positive control), spermine, spermidine or putrescine produced nociception with ED(50) of 0.16 (0.07-0.39)nmol/paw, 0.4 (0.2-0.7) μmol/paw, 0.3 (0.1-0.9) μmol/paw and 3.2 (0.9-11.5) μmol/paw, respectively. The antagonists of NMDA (MK801, 1 nmol/paw), AMPA/kainate (DNQX, 1 nmol/paw) or ASIC receptors (amiloride, 100 nmol/paw) failed to reduce the spermine-trigged nociception. However, the TRPV1 antagonists capsazepine or SB366791 (1 nmol/paw) reduced spermine-induced nociception, with inhibition of 81 ± 10 and 68 ± 9%, respectively. The previous desensitization with resiniferatoxin (RTX) largely reduced the spermine-induced nociception and TRPV1 expression in the sciatic nerve, with reductions of 82 ± 9% and 67 ± 11%, respectively. Furthermore, the combination of spermine (100 nmol/paw) and RTX (0.005 fmol/paw), in doses which alone were not capable of inducing nociception, produced nociceptive behaviors. Moreover, different concentrations of spermine (3-300 μM) enhanced the specific binding of [(3)H]-RTX to TRPV1 receptor. Altogether, polyamines produce spontaneous nociceptive effect through the stimulation of TRPV1, but not of ionotropic glutamate or ASIC receptors.