[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.
PLoS ONE 07/2015; 10(7):e0130809. DOI:10.1371/journal.pone.0130809 · 3.23 Impact Factor
[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.
Mediators of Inflammation 05/2015; 2015(Article ID 138461):9. DOI:10.1155/2015/138461 · 3.24 Impact Factor
[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.
International Journal of Molecular Sciences 03/2015; 16(4):7027-7044. DOI:10.3390/ijms16047027 · 2.86 Impact Factor
[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.
PLoS ONE 11/2014; 9(11):e110945. DOI:10.1371/journal.pone.0110945 · 3.23 Impact Factor
[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.
[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.
Microvascular Research 07/2014; 95. DOI:10.1016/j.mvr.2014.07.002 · 2.43 Impact Factor
[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).
BMC Complementary and Alternative Medicine 05/2014; 14(1):177-177. DOI:10.1186/1472-6882-14-177 · 1.88 Impact Factor
[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.
Microvascular Research 05/2014; DOI:10.1016/j.mvr.2014.02.011 · 2.43 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: Inflammatory cells surround breast carcinomas and may act promoting tumor development or stimulating anti-tumor immunity. N-acetylglucosaminidase (NAG) has been employed to detect macrophage accumulation/activation. Myeloperoxidase (MPO) is considered a marker for neutrophils activity/accumulation. Vascular Endothelial Growth Factor (VEGF) is as strong pro-angiogenic cytokine. The aim of this study was to measure the systemic inflammatory response by measuring serum levels of NAG, MPO and VEGF in women diagnosed with breast cancer and associate this response to the peritumoral inflammatory infiltrate and to prognostic factors. Serum samples obtained from women with no evidence of disease (n=31) and with breast cancer (n=68) were analyzed for the activities of NAG, MPO and VEGF by enzymatic assay. Serum levels of NAG and VEGF were higher in healthy volunteers (P<0.0001) and serum levels of MPO were higher in patients with breast cancer (P=0.002). Serum levels of NAG were positively correlated to serum levels of MPO and VEGF (P<0.0001 and P=0.0012, respectively) and MPO and VEGF serum levels had also a positive correlation (P=0.0018). The inflammatory infiltrate was not associated to serum levels of the inflammatory markers, and higher levels of MPO were associated to lymphovascular invasion negativity (P=0.0175).
[Show abstract][Hide abstract] ABSTRACT: Carboplatin, efficient cytostatics for cancer therapy, could induce apoptosis and inhibit the growth of vascular endothelium in several tumor cell lines and xenograft models. It has been suggested that the antitumor effect of chemotherapy could be increased by combining it with an antiangiogenesis agent in anticancer strategy. The present study explored the potential to increase the antitumor effect of carboplatin by combining it with thalidomide in mouse 4T1 breast cancer models, and the underlining mechanism was investigated. The systemic administration of carboplatin and thalidomide significantly decreased tumor growth through increased tumor cell apoptosis compared with either control group. Collectively, these findings suggest that combined treatment has shown synergistic suppression in tumor progression according to the analysis. Furthermore, also was observed reduction in number of lung metastases as compared to isolated treatments and increased survival of the animals. The present study may be important in future exploration of the potential application of the combined approach in the treatment of breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Introduction: The aim of this study was to investigate the value of placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and sFlt-1/PLGF ratio, in predicting symptomatic preeclampsia (PE). Methods: A prospective longitudinal study was carried out on 71 high risk preeclamptic women cohort. All of them had normal blood pressure level (≤140/90 mmHg) at the time of enrolment, 26.8 ± 1.5 weeks. Maternal blood was collected and plasma was stored in a freezer at -80 °C. PE was defined according to the National High Blood Pressure Education Program Working Group Criteria. Accuracy of angiogenic factors in predicting PE was evaluated using Receiver-operating characteristics. Results: Maternal plasma concentrations of PLGF and sFlt-1 were able to predict PE (0.90, p < 001; 0.78, p = 0.003, area under the curve, respectively) but the sFlt-1/PLGF ratio presented the best prediction potential over the others (0.95, area under the curve, p < 0.001). Conclusion: All angiogenesis factors were effective biomarkers in predicting PE during the second trimester, before the clinical onset of PE.
Hypertension in Pregnancy 08/2013; 32(3):312-20. DOI:10.3109/10641955.2013.807818 · 1.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: Angiogenesis depends on a complex interaction between cellular networks and mediators. The endocannabinoid system and its receptors have been shown to play a role in models of inflammation. Here, we investigated whether blockade of cannabinoid receptors may interfere with inflammatory angiogenesis. MATERIALS AND METHODS: Polyester-polyurethane sponges were implanted in C57Bl/6j mice. Animals received doses (3 and 10 mg/kg/daily, s.c.) of the cannabinoid receptor antagonists SR141716A (CB1) or SR144528 (CB2). Implants were collected at days 7 and 14 for cytokines, hemoglobin, myeloperoxidase, and N-acetylglucosaminidase measurements, as indices of inflammation, angiogenesis, neutrophil and macrophage accumulation, respectively. Histological and morphometric analysis were also performed. RESULTS: Cannabinoid receptors expression in implants was detected from day 4 after implantation. Treatment with CB1 or CB2 receptor antagonists reduced cellular influx into sponges at days 7 and 14 after implantation, although CB1 receptor antagonist were more effective at blocking leukocyte accumulation. There was a reduction in TNF-α, VEGF, CXCL1/KC, CCL2/JE, and CCL3/MIP-1α levels, with increase in CCL5/RANTES. Both treatments reduced neovascularization. Dual blockade of cannabinoid receptors resulted in maximum inhibition of inflammatory angiogenesis. CONCLUSIONS: Blockade of cannabinoid receptors reduced leukocyte accumulation, inflammation and neovascularization, suggesting an important role of endocannabinoids in sponge-induced inflammatory angiogenesis both via CB1 and CB2 receptors.
Inflammation Research 05/2013; DOI:10.1007/s00011-013-0638-8 · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have been shown to ameliorate a number of vascular diseases. We evaluated the inflammatory and angiogenic components of the fibrovascular tissue induced by subcutaneous implants in mice and their modulation by fluvastatin. Our results showed that the statin (0.6 and 6 mg/kg/day) inhibited hemoglobin (Hb) content (51%) and vascular endothelial growth factor (VEGF) levels (71%) in the treated group compared with the control group. The inflammatory component, as assessed by N-acetyl-β-D-glucosaminidase activity and tumor necrosis factor-α (TNF-α) level was also decreased by the compound. In the treated group; the inhibition of the enzyme activity was 33% and the cytokine was 67% relative to the control. In these implants the statin was also able to decrease nitric oxide (NO) production, detected with an NO-sensitive electrode. To our knowledge this is the first study demonstrating an inhibitory role of fluvastatin on the production of NO in inflammatory angiogenesis of newly formed fibrovascular tissue.