Effects of concentrated growth factors on the angiogenic properties of dental pulp cells and endothelial cells: an in vitro study

Abstract The aim was to investigate the angiogenic effects of concentrated growth factors on human dental pulp cells and human umbilical vein endothelial cells. Cells were treated with concentrated growth factor extracts. The CCK-8 assay and cell cycle assay were conducted to evaluate cell growth. Cell migration was evaluated by the Transwell migration assay. Angiogenesis-associated mRNA and protein expression levels were determined using quantitative real-time PCR and Western blotting, respectively. A tube formation assay was conducted to evaluate the angiogenic capacity in vitro. The data showed that compared with the control, concentrated growth factor extracts significantly promoted dental pulp cell proliferation and differentiation and endothelial cell proliferation and migration in a dose-dependent manner (p < 0.05). Concentrated growth factor extracts also promoted the tube-like structure formation of endothelial cells in vitro. The RT-PCR and Western blot results showed that concentrated growth factor extracts upregulated the expression of angiogenesis-related genes - chemokine receptor-4, platelet-derived growth factor, and vascular endothelial growth factor - in dental pulp cells. In conclusion, concentrated growth factors showed proangiogenic effects on dental pulp cells and endothelial cells and have good application potential for dental pulp revascularization.

Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns / Análise do perfil de citocinas e fatores de crescimento em plasma rico em plaquetas obtido por meio das metodologias do sistema aberto e colunas

ABSTRACT Objective: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods. Methods: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1). Results: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1β. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-β1 in relation to other systems and low FGF-2 levels. Conclusion: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used.

RESUMO Objetivo: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação. Métodos: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-β1). Resultados: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1β. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-β1 em relação aos demais sistemas e reduzidos níveis de FGF-2. Conclusão: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.

Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2. 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 mol.L1 (S. aureus) and 10.10 mol.L1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 g of LyeTx I-K-HYNIC; 250 g of SnCl2. 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n= 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.

Effect of vaginally administered fumagillin on immunohistochemical distribution of leukemia inhibitory factor, interleukin 6, transforming growth factor beta and vascular endothelial growth factor in implantation stage endometrium of the rhesus monkey.

Intravaginal administration of an anti-angiogenic agent, fumagillin, during blastocyst implantation inhibits pregnancy establishment in a dose-related manner in the rhesus monkey. In the present study, mated female rhesus monkeys were vaginally inserted with tampons containing vehicle (group 1; n = 5) and test agent (fumagillin, 4 mg/animal; group 2; n = 6) on cycle day 20, and endometrial tissue samples were collected on cycle day 24 from all monkeys and processed for histological examination and immunohistochemical localization for LIF, IL-6, TGF-beta and VEGF. Concentrations of estradiol-17 beta, progesterone and chorionic gonadotrophin in peripheral circulation were determined. From the serum profiles of the hormones, 2 monkeys in group 1, and 1 monkey in group 2 appeared pregnant. However, endometrial morphology revealed histological evidence of pregnancy in 3 out of 6 fumagillin-treated animals. Histometric analysis of immunohistochemical staining in epithelial, stromal and vascular compartments revealed that per cent areas occupied by immunoprecipitate for the cytokines studies did not change in epithelial and stromal compartments, except that for TGF-beta which was higher (P < 0.05) in epithelial compartment in group 2. No change was observed in immunoprecipitation areas for IL-6 in epithelial, stromal and vascular compartments. On the other hand, changes (P < 0.05) for LIF, TGF-beta and VEGF were evident in the vascular compartment. It is possible that disparate responses observed in glandular, stromal and vascular compartments in implantation stage endometrium following fumagillin treatment actually caused from associated decline in progesterone concentration in peripheral circulation. It is also possible that fumagillin, an angiostatic agent, affects the synthesis and secretion of cytokines primarily in the vascular compartment of implantation stage endometrium, and thereby manifests differential responses in epithelial, stromal and vascular compartments.

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart