[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to evaluate the in vivo anti-inflammatory angiogenesis activity and in vitro cytotoxicity on normal and cancer cell models of a drug delivery system consisting of poly(lactic-co-glycolic acid) nanofibers loaded with daunorubicin (PLGA-DNR) that were fabricated using an electrospinning process. The PLGA-DNR nanofibers were also characterized by thermogravimetric analysis (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM) and confocal fluorescence microscopy. In vitro release of DNR from the nanofibers and its corresponding mechanism were also evaluated. Sixty-five percent of the DNR was released in an initial burst over 8h, and by 1224h, eighty-five percent of the DNR had been released. The Higuchi model yielded the best fit to the DNR release profile over the first 8h, and the corresponding data from 24 to 1224h could be modeled using zero-order kinetics. The PLGA-DNR nanofibers exhibited a higher cytotoxicity to A431 cells than free DNR but a cytotoxicity similar to free DNR against fibroblast cells. A higher antiangiogenic effect of PLGA nanofibers was observed in the in vivo data when compared to free DNR, and no inflammatory potential was observed for the nanofibers.
[Show abstract][Hide abstract] ABSTRACT: Erlotinib (ERL) is a drug used in epidermoid carcinoma treatment. One of the ERL drawbacks is low water solubility, which limits its use in the development of safer and effective formulations. The present study used the strategy of inclusion complexation with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to increase the ERL water solubility, characterize the inclusion complex physico-chemically, and evaluate the in vitro cytotoxicity and in vivo antiangiogenic effect. The data showed 1:1 molar ratio ERL:HP-beta-CD inclusion complex formation both in solution and in solid state. Phase-solubility analysis showed A(L)-type diagrams. Isothermal titrations calorimetry and nuclear Overhauser effect spectroscopy also support that formation. Despite free ERL higher cytotoxicity, higher values were associated with the complex compared with free ERL (37.5 A mu M), and the complex was more cytotoxic to A431 human epidermoid carcinoma cell than to osteoblasts (non-cancerous cells). In addition, the inclusion complex exhibited antiangiogenic activity without affecting the activation and recruitment of neutrophils and macrophage. Overall, these results suggest that the ERL:HP-beta-CD inclusion complex could be a promising approach for developing safe and effective ERL formulation by different routes of administration.
No preview · Article · Sep 2015 · Journal of inclusion phenomena and macrocyclic chemistry
[Show abstract][Hide abstract] ABSTRACT: Based on the notion that inflammation favors tumorigenesis, our experiments comparatively assessed the influence of acute and chronic inflammation on the development of a murine mammary tumor (4T1). In addition, we characterized angiogenic and inflammatory markers in the tumor tissue and systemically. Subcutaneous implantation of polyether-polyurethane sponge discs in Balb/c mice was used to host 4T1 tumor cells (1x106), which were inoculated intraimplant 24h or 10 days post implantation. Flow cytometric analysis of enzyme-digested implants revealed that, after 24 hours, the population of leukocytes was primarily characterized by neutrophils (42.53% +/- 8.45) and monocytes (37.53% +/- 7.48), with some lymphocytes (16.27% +/- 4.0) and a few dendritic cells (1.82% +/- 0.36). At 10 days, macrophages were predominant (37.10% +/- 4.54), followed by lymphocytes (28.1% +/- 4.77), and monocytes (22.33% +/- 3.05), with some dendritic cells (13.60% +/- 0.55) and neutrophils (11.07% +/- 2.27). A mammary tumor grown in a chronic inflammatory environment was 2-fold when compared with one grown in acute inflammation and 5-fold when compared with tumor alone. The levels of pro-angiogenic cytokine (VEGF-Vascular Endothelial Growth Factor) were higher in implant-bearing tumor when 4T1 cells were grown in 10-day old implants as compared to the VEGF levels of the two other groups. Overall, the levels of the inflammatory markers evaluated (NAG -N-acetylglucosaminidase, TNF-α -Tumor Necrosis Factor- α) were higher in both groups of implant-bearing tumors and in serum from those animals when compared with the tumor alone levels. This inflammation-related difference in tumor growth may provide new insights into the contribution of different inflammatory cell populations to cancer progression.
[Show abstract][Hide abstract] ABSTRACT: There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS −/−) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS −/− mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS −/− mice. In contrast, the iNOS −/− implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS −/− mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice.
Full-text · Article · May 2015 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases OPEN ACCESS Int. J. Mol. Sci. 2015, 16 7028 act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-β displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.
Full-text · Article · Mar 2015 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: Inflammation, angiogenesis and fibrogenesis are key components of physiological (wound repair) and pathological conditions (rheumatoid arthritis, psoriasis, cancer). These processes can be induced using implantation of biomaterials allowing, therefore the characterization of cellular and molecular events involved in the development of the fibrovascular tissue. Damage to tissue is resolved through a series of overlapping phases (inflammation, cell proliferation/migration and tissue remodeling) leading to variable outcomes depending on various systemic and/or local factors. The implantation technique has been used for the assessment of the various phases of normal healing as well as the foreign body reaction. This approach has characterized the mechanisms regulating the response to injury represented by the implant and the influence of a number of factors (pathological conditions, genetic manipulation). In addition, modulation of the process by potential therapeutic compounds has been extensively studied using synthetic matrix-based methodology. We provide here a detailed description of the methodology.
[Show abstract][Hide abstract] ABSTRACT: Integrins are involved in a number of physio-pathological processes including wound healing, chronic inflammation and neoplasias. Blocking its activity is potentially of therapeutic value in these conditions. We investigated whether DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom, could modulate key events (inflammatory cell recruitment/activation, neovascularization and extracellular matrix deposition) of the proliferative fibrovascular tissue induced by polyether polyurethane sponge implants in mice. The hemoglobin content (μg/mg wet tissue), blood flow measurements (laser Doppler perfusion imaging) and number of vessels in the implants, used as indices of vascularization, showed that the disintegrin dose-dependently reduced angiogenesis in the implants relative to the Saline-treated group. DisBa-01 inhibited neutrophil and macrophage content as determined by the myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activities, respectively. Similarly, down regulation of the fibrogenic component studied (collagen deposition) was observed in DisBa-01-treated implants. VEGF, bFGF, TNF-α, CXCL1 and CCL2 levels were also decreased by the disintegrin. The inhibitory effect of this αvβ3-blocking disintegrin on the angiogenic, inflammatory, and fibrogenic components of the fibrovascular tissue induced by the synthetic matrix extends the range of DisBa-01 actions and may indicate its therapeutic potential in controlling angiogenesis in fibroproliferative diseases.
[Show abstract][Hide abstract] ABSTRACT: Implantation of synthetic matrices and biomedical devices in diabetic individuals has become a common procedure to repair and/or replace biological tissues. However, an adverse foreign body reaction that invariably occurs adjacent to implant devices impairing their function is poorly characterized in the diabetic environment. We investigated the influence of this condition on the abnormal tissue healing response in implants placed subcutaneously in normoglycemic and streptozotocin-induced diabetes in rats. In polyether-polyurethane sponge discs removed 10 days after implantation, the components of the fibrovascular tissue (angiogenesis, inflammation, fibrogenesis, and apoptosis) were assessed. Intra-implant levels of hemoglobin and vascular endothelial growth factor were not different after diabetes when compared with normoglycemic counterparts. However, there were a lower number of vessels in the fibrovascular tissue from diabetic rats when compared with vessel numbers in implants from non-diabetic animals. Overall, the inflammatory parameters (neutrophil accumulation - myeloperoxidase activity, tumor necrosis factor alpha, and monocyte chemotactic protein-1 levels and mast cell counting) increased in subcutaneous implants after diabetes induction. However, macrophage activation (N-acetyl-β-D-glucosaminidase activity) was lower in implants from diabetic rats when compared with those from normoglycemic animals. All fibrogenic markers (transforming growth factor beta 1 levels, collagen deposition, fibrous capsule thickness, and foreign body giant cells) decreased after diabetes, whereas apoptosis (TUNEL) increased. Our results showing that hyperglycemia down regulates the main features of the foreign body reaction induced by subcutaneous implants in rats may be relevant in understanding biomaterial integration and performance in diabetes.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the presence of myeloperoxidase (MPO), N-acetyl-β-D-glucosaminidase (NAG), tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF) in peripheral and menstrual blood in women with (n = 10) and without (n = 7) endometriosis. NAG and MPO activities were evaluated by enzymatic methods, whereas TNF-α and VEGF by immunoassay. No significant differences were found for these markers, neither in menstrual nor in peripheral blood between groups. Menstrual blood NAG (P = 0.039) and MPO (P = 0.0117) activities in the endometriosis group were significantly higher than in peripheral blood. NAG and MPO presented positive linear correlation in peripheral (P = 0.07; r = 0.641) and menstrual blood (P = 0.01; r = 0.603). These findings point to the existence of an increased local inflammatory activity in women with endometriosis.
Full-text · Article · Aug 2014 · Biomedecine [?] Pharmacotherapy
[Show abstract][Hide abstract] ABSTRACT: Subcutaneous implantation of synthetic materials and biomedical devices often induces abnormal tissue healing - the foreign body reaction - which impairs their function. Here we investigated the role of the chemokine receptor CCR2 in this reaction to subcutaneous implants in mice. We measured angiogenesis, inflammation and fibrogenesis induced by implantation, for 1, 4, 7 and 14days, of polyether-polyurethane sponges in mice with genetic deletion of CCR2 (KO) and WT mice. Blood flow was determined by dye diffusion and laser Doppler perfusion techniques. Cytokines (VEGF, TNF-α, CCL2, TGF-β1) were measured by ELISA. Histochemical methods were used to assess collagen deposition and macrophage-derived giant cells in the implants. Skin and implant blood flow were lower in CCR2 KO than in WT mice, as were other aspects of neo-vascularization of the implants. Neutrophil accumulation was increased in KO implants but macrophage accumulation was decreased. Implant content of CCL2 was higher in KO implants, but TGF-β1, collagen deposition and the number of foreign body giant cells were lower than in WT implants. Deletion of CCR2 decreased blood flow in normal skin and inhibited neo-vascularization, chronic inflammation and fibrogenesis in subcutaneous implants. The chemokine receptor CCR2 plays an important role in both normal skin and in the reaction elicited by subcutaneous implantation of a foreign body.
No preview · Article · Jul 2014 · Microvascular Research
[Show abstract][Hide abstract] ABSTRACT: Envenomation by the Loxosceles spider causes loxoscelism, a pattern of signs and symptoms that primarily manifests in the dermonecrotic form. Our studies have shown that a mouse subcutaneous sponge implantation model may be useful in evaluating the effects of Loxosceles similis venom. This model provides an ideal microenvironment in which to study loxoscelism; however, it is still important to evaluate its pathogenesis and to observe the effects of L. similis venom for longer time periods than those in previous studies of this model. The aims of this study are: (1) to histologically characterize the effects of L. similis crude venom in a subcutaneous sponge implant; (2) to quantify the mast cells present in the implant and to measure their degranulation activity; (3) to quantify collagen subtypes I and III; and (4) to verify, quantify, and evaluate the effects of apoptosis in the implant on the pathogenesis of loxoscelism at 1h, 4h, and 24 h after injecting the venom. Thirty Swiss mice (6-8 weeks old, male) were subcutaneously implanted with polyester-polyurethane sponge discs. Fourteen days post-implantation, the animals were divided into six groups (5 animals per group): three control groups (C1h, C4h, and C24h), in which the mice received 30 μl injections of intra-implant saline, and three treated groups (T1h, T4h, and T24h), in which the mice received 30 μL (0.5 μg) injections of L. similis crude venom at 1h, 4h, and 24 h intervals. After each time interval, the animals were euthanized, and the implants were harvested and processed for light and electron microscopic analyses. The following results were observed in the implants harvested from the treated groups: acute inflammation with marked edema, thrombus, and vasculitis, as well as increased levels of mast cells and mast cell degranulation, and apoptosis in giant cells. Furthermore, degradation of collagen types I and III was observed. An analysis of the ultrastructure revealed apoptosis in various cell types. The present results suggest that apoptosis in some cell types associated with an increase in mast cell degranulation and the degradation of collagen fibers are important in the pathogenesis of loxoscelism therefore may explain the difficulty in repairing the ulcer is commonly observed in severe cases of loxoscelism cutaneous in humans.
[Show abstract][Hide abstract] ABSTRACT: Background
Chronic inflammatory processes in the peritoneal cavity develop as a result of ischemia, foreign body reaction, and trauma. Brazilian green propolis, a beeswax product, has been shown to exhibit multiple actions on inflammation and tissue repair. Our aim was to investigate the effects of this natural product on the inflammatory, angiogenic, and fibrogenic components of the peritoneal fibroproliferative tissue induced by a synthetic matrix.
Chronic inflammation was induced by placing polyether-polyurethane sponge discs in the abdominal cavity of anesthetized Swiss mice. Oral administration of propolis (500/mg/kg/day) by gavage started 24 hours after injury for four days. The effect of propolis on peritoneal permeability was evaluated through fluorescein diffusion rate 4 days post implantation. The effects of propolis on the inflammatory (myeloperoxidase and n-acetyl-β-D-glucosaminidase activities and TNF-α levels), angiogenic (hemoglobin content-Hb), and fibrogenic (TGF-β1 and collagen deposition) components of the fibrovascular tissue in the implants were determined 5 days after the injury.
Propolis was able to decrease intraperitoneal permeability. The time taken for fluorescence to peak in the systemic circulation was 20 ± 1 min in the treated group in contrast with 15 ± 1 min in the control group. In addition, the treatment was shown to down-regulate angiogenesis (Hb content) and fibrosis by decreasing TGF-β1 levels and collagen deposition in fibroproliferative tissue induced by the synthetic implants. Conversely, the treatment up-regulated inflammatory enzyme activities, TNF-α levels and gene expression of NOS2 and IFN-γ (23 and 7 fold, respectively), and of FIZZ1 and YM1 (8 and 2 fold) when compared with the untreated group.
These observations show for the first time the effects of propolis modulating intraperitoneal inflammatory angiogenesis in mice and disclose important action mechanisms of the compound (downregulation of angiogenic components and activation of murine macrophage pathways).
Preview · Article · May 2014 · BMC Complementary and Alternative Medicine
[Show abstract][Hide abstract] ABSTRACT: Natriuretic peptide receptor-C (NPR-C) activation by C-ANP4-23, a specific agonist for this receptor, has been shown to inhibit key events of the angiogenic cascade (migration, proliferation, and vascular endothelial growth factor—VEGF—production). We investigated whether C-ANP4-23 could also inhibit angiogenesis in the sponge model in vivo. In this study, we evaluated the effects of this peptide on inflammatory and angiogenic components of the fibrovascular tissue induced by polyether polyurethane sponge implants in mice. The hemoglobin content (μg/mg wet tissue) and blood flow measurements (laser Doppler perfusion imaging) of the implants, used as an index of vascularization, showed that single or multiple doses of C-ANP4-23 reduced angiogenesis in the implants relative to the PBS-treated group. The peptide exerted an inhibitory effect on nitric oxide production (nitrite levels) and a dual effect on VEGF levels, depending on the number of doses. Histological analysis corroborated the biochemical and functional parameters indicative of neovascularization inhibition (decreased vessel number). The peptide failed to modulate inflammation in our system. The inhibitory effect of C-ANP4-23 on the angiogenic component of the fibrovascular tissue induced by the synthetic matrix extends the range of the peptide's actions and may indicate its therapeutic potential in controlling angiogenesis in fibroproliferative diseases.This article is protected by copyright. All rights reserved.
No preview · Article · May 2014 · Clinical and Experimental Pharmacology and Physiology
[Show abstract][Hide abstract] ABSTRACT: The increased prevalence of diabetes worldwide is associated with increasing numbers of diabetic individuals receiving synthetic matrices and biomedical implants to repair and/or replace biological tissues. This therapeutic procedure invariably leads to adverse tissue healing (foreign body reaction), thus impairing the biomedical device function of subcutaneous implants. However, the influence of diabetes on abnormal tissue healing in intraperitoneal implants is unclear. We investigated key components of foreign body reactions in diabetic rats. Polyether-polyurethane sponge discs were placed intraperitoneally in rats previously injected with streptozotocin for induction of diabetes and in non-diabetic rats. Implants removed 10 days after implantation were assessed by determining of the components of the fibrovascular tissue (angiogenesis, inflammation, and fibrogenesis). In implants from diabetic rats, fibrous capsule thickness and fibrovascular tissue infiltration (hematoxilin & eosin and picrossirius staining) were reduced in comparison with implants from non-diabetic rats. Hemoglobin (Hb) content (vascular index) and VEGF levels (pro-angiogenic cytokine) were increased after diabetes. However, the number of vessels (H&E and CD31-immunostaining) in the fibrovascular tissue from diabetic rats was decreased when compared with vessel numbers in implants from non-diabetic animals. Overall, all inflammatory parameters (macrophage accumulation-NAG activity; TNF-α and MCP-1 levels) increased in intraperitoneal implants after diabetes induction. The pro-fibrogenic cytokine (TGFβ-1) increased after diabetes, but collagen deposition remained unaltered in the implants from diabetic rats. These important diabetes-related changes (increased levels of pro-inflammatory and angiogenic and fibrogenic cytokines) in peritoneal implant healing provide an insight into the mechanisms of the foreign body response in the diabetic environment in rats.
Full-text · Article · May 2014 · Microvascular Research
[Show abstract][Hide abstract] ABSTRACT: Fibroproliferative processes are regulated by a wide variety of tissue components and genetic factors. However, whether there are genetic differences in peritoneal fibroproliferative tissue formation, with consequent differences in response to drug treatment, is unclear. We characterize the influence of the genetic background on peritoneal fibroproliferative tissue induced by sponge implants in DBA/1, Swiss, C57BL/6, and BALB/c mouse strains. In addition, responses to dipyridamole in the implants were evaluated. Angiogenesis, assessed by intra-implant hemoglobin content, was highest in Swiss mice, whereas levels of vascular endothelial growth factor were highest in C57BL/6 mice. The levels of pro-inflammatory cytokines and of inflammatory enzymes (myeloperoxidase- and N-acetyl-β-D-glucosaminidase) were also strain-related. The pro-fibrogenic markers transforming growth factor beta-1 and collagen were lowest in implants placed in DBA/1 mice, whereas those in C57BL/6 mice had the highest levels. Differential sensitivity to dipyridamole was also observed, with this compound being pro-angiogenic in implants placed in DBA/1 mice but antiangiogenic in implants placed in Swiss. An overall anti-inflammatory response was observed in the inbred strains. Antifibrogenic effects were observed only in implants placed in C57BL/6 mice. These important strain-related differences in the development of peritoneal fibrosis and in response to dipyridamole must be considered in the design and analysis of studies on fibrogenesis in mice.
Full-text · Article · May 2014 · Wound Repair and Regeneration