Pollyana Ribeiro Castro

Genetic background determines mouse strain differences in inflammatory angiogenesis.

Inflammation and angiogenesis are key components of fibrovascular tissue growth, a biological event underlying both physiological (wound healing) and pathological conditions (tumor development, chronic inflammation). We investigated these components in three frequently used mouse strains (Swiss, Balb/c and C57BL/6J) to verify the influence of genetic background on the kinetics of inflammatory cell recruitment/activation, neovascularization, extracellular matrix deposition, and cytokine production in polyether-polyurethane sponge implanted subcutaneously in male mice of these strains. The kinetics of neutrophil recruitment/activation as assessed by myeloperoxidase (MPO) activity was 2- and 3-fold higher in Balb/c implants at day 1 compared with Swiss and C57BL/6J implants, respectively. Macrophage accumulation/activation as NAG (n-acetyl β-glucosaminidase) activity was higher in Swiss implants. The levels the monocyte chemoattractant protein 1 (CCL2(MCP-1)) peaked at day 10 in the three types of implants but was produced more by C57BL/6J mice. Angiogenesis (hemoglobin, vascular endothelial growth factor-VEGF, and number of vessels) differed among the strains. Swiss implants had the highest hemoglobin content but the lowest VEGF levels. In contrast, Balb/c implants had higher VEGF levels but lower hemoglobin. Collagen deposition and transforming growth factor β-1; TGFβ-1 levels also varied among the groups. Swiss and Balb/c implants had progressive increase in TGFβ-1 from 4 to 14 days, while C57BL/6J implants achieved the peak at day 10 and fell at day 14. These findings emphasize the major contribution of genetic background in the temporal pattern and intensity of inflammatory angiogenesis components that may have functional consequences in physiological and pathological conditions where these processes co-exist.

Kinetics of implant-induced inflammatory angiogenesis in abdominal muscle wall in mice.

Injury of skeletal abdominal muscle wall is a common medical condition and implantation of synthetic or biological material is a procedure to repair musculofascial defects. We proposed to characterize the dynamics of inflammatory cell recruitment, newly formed blood vessels, cytokine production and fibrogenesis in the abdominal skeletal muscle in response to polyether-polyurethane sponge implants in mice. At 2, 4, 7 and 10days after implantation the muscle tissue underneath the sponge matrix was removed for the assessment of the angiogenic response (hemoglobin content, vascular endothelial growth factor and morphometric analysis of the number of vessels) and inflammation (myeloperoxidase and n-acethyl-B-d-glucosaminidase activities, cytokines). In addition, muscle fibrogenesis was determined by the levels of TGF-β1 and collagen deposition. Hemoglobin content, wash out rate of sodium fluorescein (indicative of blood flow) and the number of vessels increased in the abdominal muscle bearing the synthetic matrix in comparison with the intact muscle. Neutrophil recruitment peaked in the muscle at day 2, followed by macrophage accumulation at day 4 post-injury. The levels of the cytokines, VEGF, TNF-α, CCL-2/MCP-1 were higher in the injured muscle compared with the intact muscle and peaked soon after muscle injury (days 2 to 4). Collagen levels were higher in sponge-bearing muscle compared with the non-bearing tissue soon after injury (day 2). The implantation technique together with the inflammatory and vascular parameters used in this study revealed inflammatory, angiogenic and fibrogenic events and mechanisms associated with skeletal muscle responses to synthetic implanted materials.

Deletion of the chemokine receptor CCR2 attenuates foreign body reaction to implants in mice

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 was 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.

Differential Contribution of Acute and Chronic Inflammation to the Development of Murine Mammary 4T1 Tumors

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.

Genetic strain differences in the development of peritoneal fibroproliferative processes in mice.

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.

Alimentação complementar em crianças no segundo ano de vida

OBJETIVO: Estudar as praticas alimentares de criancas no segundo ano de vida, comparando as que estao em aleitamento materno complementado com aquelas desmamadas antes dos 12 meses de vida. METODOS: Estudo transversal envolvendo criancas de 12 a 24 meses da area de abrangencia de um servico de atencao primaria de Belo Horizonte, Minas Gerais. As maes foram entrevistadas sobre as praticas de alimentacao de seus filhos. Foram comparadas as praticas alimentares das criancas em aleitamento materno complementado com aquelas desmamadas antes dos 12 meses de vida por meio dos testes qui-quadrado ou exato de Fisher, t de Student e Kruskal-Wallis, com nivel de significância de 5%. RESULTADOS: Foram avaliadas 118 criancas com idade media de 16,8±4,0 meses, sendo que 35% delas ainda eram amamentadas e 15,3% mantiveram aleitamento exclusivo por seis meses. Nas criancas amamentadas, a duracao mediana do aleitamento exclusivo foi de quatro meses e, nas desmamadas, dois meses (p=0,13). Em ambos os grupos houve introducao precoce de alimentos complementares, elevado consumo de alimentos industrializados, alta prevalencia de consumo diario de oleos ou gorduras (90,7%) e baixo consumo de frutas (38,1%). CONCLUSOES: Os resultados sinalizam praticas alimentares inadequadas nos lactentes, independentemente do consumo recomendado de leite materno, denotando a necessidade de aprimoramento e integracao das acoes de promocao do aleitamento materno e alimentacao saudavel nos servicos de atencao primaria a saude.

Genetic background determines inflammatory angiogenesis response to dipyridamole in mice.

Inflammation and angiogenesis, key components of fibrovascular tissue growth, exhibit considerable variability among species and strains. We investigated whether the response of inbred and outbred mice strains to dipyridamole (DP) on these processes would present similar variability. The effects of the drug on blood vessel formation, inflammatory cell recruitment, collagen deposition and cytokine production were determined on the fibroproliferative tissue induced by sponge implants in Swiss and Balb/c mice. Angiogenesis as assessed by hemoglobin (Hb) and vascular endothelial growth factor (VEGF) concentrations differed between the strains. Swiss implants had the highest Hb content but the lowest VEGF concentrations. Systemic DP treatment exerted an antiangiogenic effect on Balb/c implants but an proangiogenic effect on Swiss implants. The inflammatory enzyme activities myeloperoxidase (six-fold higher in Balb/c implants) and N-acetyl-β-d-glucosaminidase were reduced by the treatment in Balb/c implants only. Nitrite concentrations were also higher in Balb/c implants by 40% after DP treatment. Tumor necrosis factor-alpha levels were similar in the implants of both strains and were not reduced by DP. Transforming growth factor β-1 levels and collagen deposition also varied between the strains. The inbred strain had similar levels of the cytokine but implants of Swiss mice presented more collagen. DP treatment reduced collagen deposition in Balb/c implants only. Our data showing the influence of the genetic background on marked heterogeneity of inflammatory angiogenesis components and differential sensitivity to DP may provide some answers to clinical evidence for resistance to angiogenic therapy.

Low Concentration of Sodium Butyrate from Ultrabraid+NaBu suture, Promotes Angiogenesis and Tissue Remodelling in Tendon-bones Injury

Sodium butyrate (NaBu), a form of short-chain fatty acid (SCFA), acts classically as a potent anti-angiogenic agent in tumour angiogenesis models, some authors demonstrated that low concentrations of NaBu may contribute to healing of tendon-bone injury in part at least through promotion of tissue remodelling. Here, we investigated the effects of low-range concentrations of NaBu using in vitro and in vivo assays using angiogenesis as the primary outcome measure and the mechanisms through which it acts. We demonstrated that NaBu, alone or perfused from the UltraBraid+NaBu suture was pro-angiogenic at very low-range doses promoting migration, tube formation and cell invasion in bovine aortic endothelial cells (BAECs). Furthermore, cell exposure to low NaBu concentrations increased expression of proteins involved in angiogenic cell signalling, including p-PKCβ1, p-FAK, p-ERK1/2, p-NFκβ, p-PLCγ1 and p-VEGFR2. In addition, inhibitors of both VEGFR2 and PKCβ1 blocked the angiogenic response. In in vivo assays, low concentrations of NaBu induced neovascularization in sponge implants in mice, evidenced by increased numbers of vessels and haemoglobin content in these implants. The findings in this study indicate that low concentrations of NaBu could be an important compound to stimulate angiogenesis at a site where vasculature is deficient and healing is compromised.

Complementary feeding of children in the second year of life

OBJETIVO: Estudar as praticas alimentares de criancas no segundo ano de vida, comparando as que estao em aleitamento materno complementado com aquelas desmamadas antes dos 12 meses de vida. METODOS: Estudo transversal envolvendo criancas de 12 a 24 meses da area de abrangencia de um servico de atencao primaria de Belo Horizonte, Minas Gerais. As maes foram entrevistadas sobre as praticas de alimentacao de seus filhos. Foram comparadas as praticas alimentares das criancas em aleitamento materno complementado com aquelas desmamadas antes dos 12 meses de vida por meio dos testes qui-quadrado ou exato de Fisher, t de Student e Kruskal-Wallis, com nivel de significância de 5%. RESULTADOS: Foram avaliadas 118 criancas com idade media de 16,8±4,0 meses, sendo que 35% delas ainda eram amamentadas e 15,3% mantiveram aleitamento exclusivo por seis meses. Nas criancas amamentadas, a duracao mediana do aleitamento exclusivo foi de quatro meses e, nas desmamadas, dois meses (p=0,13). Em ambos os grupos houve introducao precoce de alimentos complementares, elevado consumo de alimentos industrializados, alta prevalencia de consumo diario de oleos ou gorduras (90,7%) e baixo consumo de frutas (38,1%). CONCLUSOES: Os resultados sinalizam praticas alimentares inadequadas nos lactentes, independentemente do consumo recomendado de leite materno, denotando a necessidade de aprimoramento e integracao das acoes de promocao do aleitamento materno e alimentacao saudavel nos servicos de atencao primaria a saude.

Cellular and Molecular Heterogeneity Associated with Vessel Formation Processes

The microvasculature heterogeneity is a complex subject in vascular biology. The difficulty of building a dynamic and interactive view among the microenvironments, the cellular and molecular heterogeneities, and the basic aspects of the vessel formation processes make the available knowledge largely fragmented. The neovascularisation processes, termed vasculogenesis, angiogenesis, arteriogenesis, and lymphangiogenesis, are important to the formation and proper functioning of organs and tissues both in the embryo and the postnatal period. These processes are intrinsically related to microvascular cells, such as endothelial and mural cells. These cells are able to adjust their activities in response to the metabolic and physiological requirements of the tissues, by displaying a broad plasticity that results in a significant cellular and molecular heterogeneity. In this review, we intend to approach the microvasculature heterogeneity in an integrated view considering the diversity of neovascularisation processes and the cellular and molecular heterogeneity that contribute to microcirculatory homeostasis. For that, we will cover their interactions in the different blood-organ barriers and discuss how they cooperate in an integrated regulatory network that is controlled by specific molecular signatures.