Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Possible role of annexin A1/FPR2 pathway in COX2/NLRP3 inflammasome regulation in alveolar bone cells of estrogen-deficient female rats with diabetes mellitus.

BACKGROUND: Annexin A1 (ANXA1) and the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome play important roles in bone remodeling. However, expression profiles of these factors in bone cells under diabetes mellitus (DM) and estrogen-deficient conditions are poorly understood. This study investigated the immunoexpression of ANXA1 and its formyl peptide receptor 2 (FPR2), as well as NLRP3 inflammasome mediators, during remodeling of the alveolar process in diabetic and estrogen-deficient rats. METHODS: Twenty adult female Wistar rats were divided into four groups (n = 5): Sham-operated (SHAM) and ovariectomized (OVX) rats received a vehicle solution, and SHAM and OVX rats were intraperitoneally administered 60 mg/kg/body weight (BW) of streptozotocin (STZ) to induce DM (SHAM-Di and OVX-Di groups). After 7 weeks, the rats were euthanized and their maxillae were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin/eosin (H&E) and picrosirius red or subjected to immunohistochemical detection of ANXA1, FPR2, NLRP3, interleukin-1ß (IL-1ß), and cyclooxygenase-2 (COX2). RESULTS: Estrogen deficiency and DM were associated with deleterious effects in bone tissue, as evidenced by a lower number of osteocytes and higher number of empty lacunae in the SHAM-Di and OVX-Di groups compared to the nondiabetic groups. Both diabetic groups showed a smaller vascular area and weaker collagen fiber birefringence intensity in alveolar bone tissue. A significantly higher number of ANXA1/FPR2-positive osteoblasts, osteocytes, and osteoclasts was accompanied by a significantly higher number of these cells immunolabeled for COX2, NLRP3, and IL-1ß in the diabetic and OVX groups, especially in both estrogen-deficient and diabetic rats. CONCLUSION: These results indicate a possible role for the ANXA1/FPR2 pathway as a fine-tuning/anti-inflammatory regulator to counterbalance exacerbated COX2/NLRP3/IL-1ß activation in bone cells during bone remodeling under estrogen deficiency and DM.

Effects of inhibition of 5-lipoxygenase and 12-lipoxygenase pathways on skeletal muscle fiber regeneration.

We investigated the effect of inhibition of 5-lipoxigenase (LOX) and 12-LOX pathways on the regeneration of skeletal muscle fibers after injury induced by a myotoxin (MTX) phospholipase A2 from snake venom in an in vivo experimental model. Gastrocnemius muscles of mice injected with MTX presented an increase in 5-LOX protein expression, while 12-LOX was found to be a constitutive protein of skeletal muscle. Animals that received oral treatments with 5-LOX inhibitor MK886 or 12-LOX inhibitor baicalein 30 min and 48 h after MTX-induced muscle injury showed a reduction in the inflammatory process characterized by a significant decrease of cell influx and injured fibers in the degenerative phase (6 and 24 h after injury). In the beginning of the regeneration process (3 days), mice that received MK886 showed fewer new basophilic fibers, suggesting fewer proliferative events and myogenic cell fusion. Furthermore, in the progression of tissue regeneration (14-21 days), the mice treated with 5-LOX inhibitor presented a lower quantity of central nucleus fibers and small-caliber fibers, culminating in a muscle that is more resistant to the stimulus of fatigue during muscle regeneration with a predominance of slow fibers. In contrast, animals early treated with the 12-LOX inhibitor presented functional fibers with higher diameters, less resistant to fatigue and predominance of fast heavy-chain myosin fibers as observed in control animals. These effects were accompanied by an earlier expression of myogenic factor MyoD. Our results suggest that both 5-LOX and 12-LOX pathways represent potential therapeutic targets for muscle regeneration. It appears that inhibition of the 5-LOX pathway represses only the degenerative process by reducing tissue inflammation levels. Meanwhile, inhibition of the 12-LOX pathway also favors the anticipation of maturation and earlier recovery of muscle fiber activity function after injury.

Role of Annexin A1 Secreted by Neutrophils in Melanoma Metastasis.

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.

The early inhibition of the COX-2 pathway in viperid phospholipase A2-induced skeletal muscle myotoxicity accelerates the tissue regeneration.

The administration of non-steroidal anti-inflammatory drugs in the treatment of injury and muscle regeneration is still contradictory in effectiveness, especially regarding the timing of their administration. This can interfere with the production of prostaglandins originating from inflammatory isoform cyclooxygenase-2 (COX-2), which is essential to modulate tissue regeneration. The phospholipases A2 (PLA2) from viperid venoms cause myotoxicity, therefore constituting a tool for the study of supportive therapies to improve skeletal muscle regeneration. This study investigated the effect of early administration of lumiracoxib (selective inhibitor of COX-2) on the degeneration and regeneration stages of skeletal muscle after injury induced by a myotoxic PLA2. After 30 min and 48 h of intramuscular injection of PLA2, mice received lumiracoxib orally and histological, functional, and transcriptional parameters of muscle were evaluated from 6 h to 21 days. Inhibition of COX-2 in the early periods of PLA2-induced muscle degeneration reduced leukocyte influx, edema, and tissue damage. After the second administration of lumiracoxib, in regenerative stage, muscle showed increase in number of basophilic fibers, reduction in fibrosis content and advanced recovery of functionality characterized by the presence of fast type II fibers. The expression of Pax7 and myogenin were increased, indicating a great capacity for storing satellite cells and advanced mature state of tissue. Our data reveals a distinct role of COX-2-derived products during muscle degeneration and regeneration, in which early administration of lumiracoxib was a therapeutic strategy to modulate the effects of prostaglandins, providing a breakthrough in muscle tissue regeneration induced by a myotoxic PLA2.

Neutrophil depletion in the pre-implantation phase impairs pregnancy index, placenta and fetus development.

Maternal neutrophils cells are players in gestational tolerance and fetus delivery. Nonetheless, their actions in each phase of the pregnancy are unknown. We here investigated the role of maternal neutrophil depletion before the blastocyst implantation phase and outcomes in the pregnancy index, placenta, and fetus development. Neutrophils were pharmacologically depleted by i.p. injection of anti-Gr1 (anti-neutrophils; 200 µg) 24 hours after plug visualization in allogeneic-mated C57BL/6/BALB/c mice. Depletion of peripheral neutrophils lasted until 48 hours after anti-Gr1 injection (gestational day 1.5-3.5). On gestational day 5.5, neutrophil depletion impaired the blastocyst implantation, as 50% of pregnant mice presented reduced implantation sites. On gestational day 18.5, neutrophil depletion reduced the pregnancy rate and index, altered the placenta disposition in the uterine horns, and modified the structure of the placenta, detected by reduced junctional zone, associated with decreased numbers of giant trophoblast cells, spongiotrophoblast. Reduced number of placenta cells labeled for vascular endothelial growth factor (VEGF), platelet-endothelial cell adhesion molecule (PECAM-1), and intercellular cell adhesion molecule (ICAM-1), important markers of angiogenesis and adhesiveness, were detected in neutrophil depleted mice. Furthermore, neutrophil depletion promoted a higher frequency of monocytes, natural killers, and T regulatory cells, and lower frequency of cytotoxic T cells in the blood, and abnormal development of offspring. Associated data obtained herein highlight the pivotal role of neutrophils actions in the early stages of pregnancy, and address further investigations on the imbricating signaling evoked by neutrophils in the trophoblastic interaction with uterine epithelium.

Analysis of different conditioned media secreted by limbal progenitor cells in the modulation of corneal healing.

Ex vivo cultivation and transplantation of limbal epithelial cells has been reported as an alternative source for ocular surface reconstruction. However, until now, the functional improvement of these patients is limited due to the low survival rate of the transplanted cells. Consequently, the clinical benefits of this therapeutic strategy are only temporary and can assign them to paracrine effects associated with the transplanted cells. With this background in mind, we aimed to analyze the effect of different conditioned media containing growth factors secreted by limbal progenitor cells on corneal epithelial healing, both in vitro and in vivo. Limbal tissue was used to obtain different conditioned media (CM). For the in vitro assay, corneal epithelial cells were treated with CM and the epithelial migration was analyzed. Growth factors in the CM were identified with ELISA and multiplex. For the in vivo assay in rats, total limbal stem cell deficiency (LSCD) was induced with an abrasive injury to the ocular surface, and the animals were treated with different CM. Clinical and histological analyses were performed. In the in vitro assay, treatment with limbal fibroblast (LF CM) was more effective compared to the other CM, and analysis revealed high concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF). In the in vivo assay, animals treated with LF CM showed epithelial defect improvement, maintenance of thickness, and decreased opacity and neovascularization. This treatment also allowed better ocular surface tissue organization when compared to the other treatments. The in vitro and in vivo experiments showed better outcomes in the corneal wound healing for the LF CM treatment. The high concentrations of KGF and HGF, linked to epithelial cell migration and proliferation, may correlate to the best results found in this treatment.

Relationship between Papillomavirus vaccine, vaginal microbiome, and local cytokine response: an exploratory research.

INTRODUCTION: The influence of vaccination on composition of the human microbiome at distinct sites has been recognized as an essential component in the development of new vaccine strategies. The HPV vaccine is widely used to prevent cervical cancer; however, the influence of HPV vaccine on the vaginal microbiota has not been previously investigated. In his study, we performed an initial characterization of the microbiome and cytokine composition in the vagina following administration of the bivalent vaccine against HPV 16/18. MATERIAL AND METHODS: In this exploratory study, fifteen women between 18 and 40 years received three doses of the HPV-16/18 AS04-adjuvanted vaccine (Cervarix®). Cervicovaginal samples were collected before the first dose and 30 days after the third dose. HPV genotyping was performed by the XGEN Flow Chip technique. The cytokines IFN-γ, IL-2, IL-12p70, TNF-α, GM-CSF, IL-4, IL-5, IL-10, and IL-13 were quantitated by multiplex immunoassay. The vaginal microbiome was identified by analysis of the V3/V4 region of the bacterial 16S rRNA gene. RESULTS: The most abundant bacterial species in the vaginal microbiome was Lactobacillus crispatus, followed by L. iners. Bacterial diversity and dominant organisms were unchanged following vaccination. Small decreases in levels of pro and anti-inflammatory cytokines were observed following HPV vaccination, but there was no association between vaginal cytokine levels and microbiome composition. CONCLUSION: Vaginal microbiome is not altered following administration of the standard three-dose HPV-16/18 AS04-adjuvanted (Cervarix®) vaccine.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

The role of annexin A1 in Candida albicans and Candida auris infections in murine neutrophils.

Annexin A1 (AnxA1) is an anti-inflammatory protein expressed in various cell types, especially macrophages and neutrophils. Because neutrophils play important roles in infections and inflammatory processes and the relationship between AnxA1 and Candida spp. infections is not well-understood, our study examined whether AnxA1 can serve as a target protein for the regulation of the immune response during fungal infections. C57BL/6 wild-type (WT) and AnxA1 knockout (AnxA1-/-) peritoneal neutrophils were coinfected with Candida albicans or Candida auris for 4 h. AnxA1-/- neutrophils exhibited a marked increase in cyclooxygenase 2 (COX-2), phosphorylated extracellular signal-related kinase (ERK), p-38, and c-Jun N-terminal kinase (JNK) levels after coinfection with both Candida spp. A lipidomics approach showed that AnxA1 deficiency produced marked differences in the supernatant lipid profiles of both control neutrophils and neutrophils coinfected with Candida spp. compared with WT cells, especially the levels of glycerophospholipids and glycerolipids. Our results showed that endogenous AnxA1 regulates the neutrophil response under fungal infection conditions, altering lipid membrane organization and metabolism.

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia.

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17-11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

Effects of estrogen deficiency followed by streptozotocin-induced diabetes on periodontal tissues of female rats.

Although both estrogen deficiency and diabetes contribute to periodontal tissue deterioration, the combined effects of these conditions on periodontium is unknown. Thus, we analyzed the combined effects of ovariectomy followed by streptozotocin (STZ)-induced diabetes on periodontal tissues of rats. Twenty adult rats were ovariectomized (OVX) or SHAM-operated (SHAM). After 3 weeks, the rats received an intraperitoneal injection of STZ (60 mg/kg/body weight) to induce diabetes or vehicle (blank) solution. The groups were assigned as follows (n = 5): SHAM-vehicle (SHAM), OVX-vehicle (OVX), SHAM + STZ (SHAM-Di), and OVX + STZ (OVX-Di). Seven weeks post-diabetes induction, the rats were euthanized. Blood samples were collected for glucose measurements and maxillae were processed for paraffin embedding. Sections stained with hematoxylin/eosin, Masson's trichrome, and picrosirius-red were used for alveolar bone loss and collagen fiber analysis in the lamina propria. Immunohistochemistry was performed for runt-related transcription factor 2 (Runx2), matrix metalloproteinase 9 (MMP-9), and tryptase detection. Alveolar bone loss and fewer collagen fibers were observed in the OVX-Di group, collagen fibers with irregular organization, and MMP-9 immunoreactivity were more evident in diabetic groups, and MMP-9-positive osteoclasts on alveolar bone surface were noticed in all groups. The OVX-Di group showed lower Runx2 immunoreactivity (osteoblast formation marker), and more tryptase-positive cells (mast cell marker) in the alveolar bone marrow. Our results indicate that estrogen depletion, followed by STZ-induced diabetes, promotes periodontal tissue deterioration that is more evident than both interventions applied alone. Furthermore, our results points to a possible participation of bone-derived mast cells in this process.

Annexin A1 Regulates NLRP3 Inflammasome Activation and Modifies Lipid Release Profile in Isolated Peritoneal Macrophages.

Annexin A1 (AnxA1) is a potent anti-inflammatory protein that downregulates proinflammatory cytokine release. This study evaluated the role of AnxA1 in the regulation of NLRP3 inflammasome activation and lipid release by starch-elicited murine peritoneal macrophages. C57bl/6 wild-type (WT) and AnxA1-null (AnxA1-/-) mice received an intraperitoneal injection of 1.5% starch solution for macrophage recruitment. NLRP3 was activated by priming cells with lipopolysaccharide for 3 h, followed by nigericin (1 h) or ATP (30 min) incubation. As expected, nigericin and ATP administration decreased elicited peritoneal macrophage viability and induced IL-1ß release, more pronounced in the AnxA1-/- cells than in the control peritoneal macrophages. In addition, nigericin-activated AnxA1-/- macrophages showed increased levels of NLRP3, while points of co-localization of the AnxA1 protein and NLRP3 inflammasome were detected in WT cells, as demonstrated by ultrastructural analysis. The lipidomic analysis showed a pronounced release of prostaglandins in nigericin-stimulated WT peritoneal macrophages, while ceramides were detected in AnxA1-/- cell supernatants. Different eicosanoid profiles were detected for both genotypes, and our results suggest that endogenous AnxA1 regulates the NLRP3-derived IL-1ß and lipid mediator release in macrophages.

Lack of Endogenous Annexin A1 Increases Mast Cell Activation and Exacerbates Experimental Atopic Dermatitis.

Annexin A1 (AnxA1) is a protein with potent anti-inflammatory actions and an interesting target that has been poorly explored in skin inflammation. This work evaluated the lack of endogenous AnxA1 in the progression of ovalbumin (OVA)-induced atopic dermatitis (AD)-like skin lesions. OVA/Alum-immunized C57BL/6 male wild-type (WT) and AnxA1 null (AnxA1-/-) mice were challenged with drops containing OVA on days 11, 14⁻18 and 21⁻24. The AnxA1-/- AD group exhibited skin with intense erythema, erosion and dryness associated with increased skin thickness compared to the AD WT group. The lack of endogenous AnxA1 also increased IgE relative to WT animals, demonstrating exacerbation of the allergic response. Histological analysis revealed intense eosinophilia and mast-cell activation in AD animals, especially in AnxA1-/-. Both AD groups increased skin interleukin (IL)-13 levels, while IL-17A was upregulated in AnxA1-/- lymph nodes and mast cells. High levels of phosphorylated ERK were detected in keratinocytes from AD groups. However, phospho-ERK levels were higher in the AnxA1-/- when compared to the respective control groups. Our results suggest AnxA1 as an important therapeutic target for inflammatory skin diseases.

Involvement of the annexin A1-Fpr anti-inflammatory system in the ocular allergy.

Annexin A1 (ANXA1)-formyl peptide receptor (Fpr) system is potent effective mediators in the control of the inflammatory response. In this study, we evaluate the potential involvement of the Fpr family in the protective effect of the mimetic peptide of ANXA1 (ANXA12-26) using an experimental allergic conjunctivitis (AC) model in mice. Ovalbumin (OVA)/Alum-immunized wild-type (WT) and ANXA1-null (ANXA1-/-) Balb/c mice (days 0 and 7) were challenged by eye drops containing OVA on days 14-16, and two groups received ANXA12-26 alone or with Fpr antagonist Boc2 intraperitoneally during challenged days. As expected, plasma IgE anti-OVA levels increased significantly in the OVA-immunized WT and ANXA1-/- mice, supporting the efficacy of AC model. AC increased Fpr1 and Fpr2 levels in the conjunctiva and the lack of endogenous ANXA1 exacerbated Fpr2 expression only. In contrast, administering ANXA12-26 in the WT mice diminished Fpr2 levels in the conjunctiva, and the effect was reverted by Boc2. Ultrastructural analysis showed the co-localization of Fpr2 and ANXA1 in the plasma membrane of mast cells (MCs), eosinophils and neutrophils, supporting this system as being operative in the AC. Boc2 abrogated the ANXA12-26 effect by increasing the MC degranulation and the eosinophil influx in the conjunctiva, and these findings were supported by peroxidase eosinophil, eotaxin and MC protease levels. Additionally, the ANXA12-26-Fpr system in the AC was associated with the activation of ERK and JNK. Collectively, the data provided in vivo supports the anti-allergic effects of the ANXA1-Fpr system and may serve as a therapeutic target in this ocular disorder.

Annexin A12-26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes.

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.

Treatment with galectin-1 eye drops regulates mast cell degranulation and attenuates the severity of conjunctivitis.

Galectin-1 (Gal-1) is a ß-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation, however the mechanisms by which Gal-1 modulates cellular responses in allergic inflammatory processes have not been fully determined. In this study, we evaluated the therapeutic potential of Gal-1 eye drops in an experimental model of conjunctivitis. Wistar rats received a topical application of compound (C)48/80 (100 mg/ml) into right eyes and a drop of vehicle into the contralateral eye. Another group of rats received Gal-1 (0.3 or 3 µg/eye) or sodium cromoglycate (SCG; 40 mg/ml) in both eyes and, after 15 min, right eye was challenged with C48/80. Conjunctivitis-induced by C48/80 was characterized by severe eyelid oedema and tearing, but clinical signs were ameliorated by eye drop doses of both Gal-1 (0.3/3 µg) and SCG. As expected, an increased proportion of degranulated mast cells (62%, P < 0.01) and lower histamine levels were observed after 6 h of C48/80 challenge, compared to control (32%). This effect was abrogated by Gal-1 and SCG, which reduced mast cell degranulation (31-36%), eosinophil migration and eosinophil peroxidase levels in the eyes. Gal-1 (3 µg) and SCG treatments also decreased IL-4 levels, as well as activation of mitogen activated protein kinases compared to untreated C48/80 eyes. Our findings suggest that Gal-1 eye drops represent a new therapeutic strategy for ocular allergic inflammation.

Galectin-3: role in ocular allergy and potential as a predictive biomarker.

AIMS: To evaluate galectin-3 (Gal-3), a ß-galactoside binding protein, as a possible biomarker in ocular allergy and further investigated the role of endogenous Gal-3 in a murine model of ovalbumin (OVA)-induced allergic conjunctivitis (AC). METHODS: Conjunctival impression cytology specimens from control and patients with severe vernal keratoconjunctivitis, treated or untreated, were used to evaluate Gal-3 expression by immunocytochemistry. To investigate the mechanism of action of Gal-3, OVA-immunised BALB/c male wild-type (WT) and Gal-3 null (Gal-3-/-) mice were challenged with eye drops containing OVA on days 14-16 with a subset of animals pretreated with 0.03% tacrolimus (TC) or dexamethasone (Dex). RESULTS: Patients with AC and OVA-sensitised WT mice exhibited increased levels of Gal-3 in the conjunctiva compared with control, an effect reverted by the action of Dex and TC therapy. Twenty-four hours after the final OVA challenge, total and anti-OVA IgE levels increased significantly in the blood of OVA-sensitised WT and Gal-3-/- mice compared with controls, supporting the efficacy of the AC model. The lack of endogenous Gal-3 exacerbated the local inflammatory response, increasing the influx of eosinophils and mast cell activation. Additionally, OVA-sensitised Gal-3-/- animals exhibited increased CD4+ expression in the eyes as well as eotaxin, IL-4, IL-13 and interferon-γ levels in the tear fluid compared with WT animals. CONCLUSION: Gal-3 contributes to the pathogenesis of ocular allergy and represents a relevant therapeutic target.

Galectin-3 sensitized melanoma cell lines to vemurafenib (PLX4032) induced cell death through prevention of autophagy.

Melanoma is a current worldwide problem, as its incidence is increasing. In the last years, several studies have shown that melanoma cells display high levels of autophagy, a self-degradative process that can promote survival leading to drug resistance. Consequently, autophagy regulation represents a challenge for cancer therapy. Herein, we showed that galectin-3 (Gal-3), a ß-galactoside binding lectin which is often lost along melanoma progression, is a negative regulator of autophagy in melanoma cells. Our data demonstrated that Gal-3low/negative cells were more resistant to the inhibition of the activity of the cancer driver gene BRAFV600E by vemurafenib (PLX4032). Interestingly, in these cells, starvation caused further LC3-II accumulation in cells exposed to chloroquine, which inhibits the degradative step in autophagy. In addition, Gal-3 low/negative tumor cells accumulated more LC3-II than Gal-3 high tumor cells in vivo. Resistance of Gal-3low/negative cells was associated with increased production of superoxide and activation of the Endoplasmic Reticulum (ER) stress response, as evaluated by accumulation of GRP78. Pharmacological inhibition of autophagy with bafilomycin A reversed the relative resistance of Gal-3low/negative cells to vemurafenib treatment. Taken together, these results show that the autophagic flux is dependent on Gal-3 levels, which attenuate the prosurvival role of autophagy.

Evaluation of galectin-1 and galectin-3 as prospective biomarkers in keratoconus.

AIMS: To evaluate the expression of ß-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. METHODS: Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco's modified eagle medium/Ham's F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. RESULTS: Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. CONCLUSION: Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.