Regenerative effect of platelet-rich plasma in the murine ischemic limbs.

AIMS: The purpose of this study was to investigate the effect of PPRP (pure PRP) and LPRP (PRP with leukocytes) on recovery from limb ischemia and on expression of growth factors involved in angiogenesis, myogenesis and fibrogenesis. MATERIAL AND METHODS: PPRP and LPRP prepared by centrifugation were added to cultures of C2C12 and NIH3T3 cells (1 or 10% PRPs) to evaluate alterations in cell metabolism and expression of growth factors by MTT, ELISA and RT-qPCR, respectively. To evaluate in vivo regenerative effects, PRPs were injected into the ischemic limbs of BALB/c mice and muscle mass/strength and histomorphometry were evaluated after 30 days. KEY FINDINGS: Mice treated with PRPs after limb ischemia showed an increase in the size of myofibers and muscle strength, reduced fibrosis and adipocytes, and decreased capillary density and necrosis scores compared to untreated mice. In cell culture, serum deprivation reduced the viability of C2C12 and NIH3T3 cells to about 50%, but the addition of 1% PRPs completely recovered this loss. Both PRPs, downregulated most of the tested genes; however, angiogenic gene Vegfa in C2C12 and the fibrogenic genes Col1a1 and Col3a1 in NIH3T3 cells were upregulated by LPRP. SIGNIFICANCE: PPRP and LPRP had similar effects in regulation of genes involved in angiogenesis, myogenesis and fibrogenesis. However, the presence of leucocytes did not significantly affect regenerative activities of PRP in the ischemic limb.

Long-term effects for acute phase myocardial infarct VEGF165 gene transfer cardiac extracellular matrix remodeling.

BACKGROUND: Cardiac remodeling is ultimately regulated by components of the extracellular matrix (ECM). We investigated the important role that growth factors play in the regulation of ECM remodeling that occurs as a consequence of myocardium damage. METHODS AND RESULTS: Rats were submitted to the ligation of the left anterior coronary artery and pcDNA3-vascular endothelial growth factor (VEGF)(165) was immediately injected intramyocardially in the treated group. The animals were divided into large size myocardium infarction (LMI) and small size myocardium infarction, with or without gene transfer. The plasmid-containing DNA encoding VEGF(165) was injected into the cardiac muscle and its effect was observed on the ECM components. Glycosaminoglycans were identified and quantified by agarose gel based electrophoresis and ELISA as well as immunocytochemistry to examine specific cathepsin B, heparanase, and syndecan-4 changes. The amounts of hyaluronic acid (HA; p < 0.005), DS, chondroitin sulfate, and heparan sulfate (p < 0.001) were significantly increased in the LMI treated group in comparison to the other groups, which correlates with the decrease in the expression of heparanase. A decrease in the molecular mass of HA was found in the scar tissue of treated group. CONCLUSIONS: The data obtained strongly support the idea that changes in the ECM and its components are important determinants of cardiac remodeling after myocardium infarct and may be essential for inflammatory response and attempt to stabilize the damage and provide a compensatory mechanisms to maintain cardiac output since the ECM components analyzed are involved with angiogenesis, cell proliferation and differentiation.

Platelet-Rich Plasma in a Murine Model: Leukocytes, Growth Factors, Flt-1, and Muscle Healing.

BACKGROUND: It is well known that platelet-rich plasma (PRP) preparations are not the same and that not all preparations include white blood cells, but the part that leukocytes play on the healing role of PRP is still unknown. PURPOSE: The primary aim of this study was to evaluate the influence of leukocytes in different PRP preparations with a special emphasis on growth factor concentrations. The secondary aim was to evaluate the influence of PRP on muscle healing. STUDY DESIGN: Controlled laboratory study. METHODS: Two PRP preparation procedures were evaluated. Blood fractions were stained with Rapid Panoptic, and growth factors (transforming growth factor beta 1 [TGF-ß1], vascular endothelial growth factor [VEGF], insulin-like growth factor [IGF], epidermal growth factor [EGF], hepatocyte growth factor [HGF], and platelet-derived growth factor [PDGF]) were quantified by enzyme-linked immunosorbent assay. Western blotting analysis was performed for Fms-related tyrosine kinase 1 (Flt-1). A muscle contusion injury was created and treated with PRP at different time points. RESULTS: Leukocytes were the main source of VEGF, and all other growth factors measured had a higher concentration in the preparations that included the buffy coat and consequently had a higher concentration of white blood cells. Flt-1 was also found in platelet-poor plasma (PPP). There were higher concentrations of PDGF and HGF in the preparations that encompassed the buffy coat. A PRP injection 7 days after the injury provided significantly increased exercise performance and decreased the fibrotic area when compared with other PRP-treated groups. CONCLUSION: VEGF is only present in PRP's buffy coat, while Flt-1 is present in PPP. A PRP injection 7 days after an injury resulted in improved exercise performance. CLINICAL RELEVANCE: The presence of Flt-1 in PRP provides yet another explanation for results described in the literature after a PRP injection. This information is relevant for selecting the best PRP for each type of injury.

The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor.

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 µM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

[Comparison of anti-angiogenic effect in vitro between ranibizumab and bevacizumab]. / Comparação do efeito antiangiogênico do ranibizumab e do bevacizumab in vitro.

PURPOSE: To evaluate the comparative in-vitro antiangiogenic effect of Bevacizumab and Ranibizumab. METHODS: Endothelial venous umbilical cells culture (ECV304) cultivated in F12 media with addition of 10% Fetal Bovine Serum, were plaqued and treated with clinically relevant concentrations of Bevacizumab and Ranibizumab just after the scratch done in the middle of the culture (scratch methodology). Measurements of the linear size of the area free of cell proliferation were done 24, 48 and 72 hours after the scratch day point. All the experiments were done in triplicate and statistical analysis were done with T-student test. RESULTS: Inhibitory effect was observed just at the concentrations of 0.5 and 0.7 mg/ml in both drugs. At 0.7 mg/ml, Ranibizumab demonstrated a more potent proliferative inhibitory effect than Bevacizumab. At the same concentration, Ranibizumab was three times more potent than Ranibizumab. Inhibitory effect was observed just in the first 24 hours for both drugs. CONCLUSION: Ranibizumab demonstrates an increased effect when compared to Bevacizumab and this is related more to the different molar rate of each drug than related to a real better proliferative inhibitory effect.

Comparação do efeito antiangiogênico do ranibizumab e do bevacizumab in vitro / Comparison of anti-angiogenic effect in vitro between ranibizumab and bevacizumab

Objetivo: Comparar o efeito anti-angiogênico in vitro do Bevacizumab e do Rani bizumab. Métodos: Células endotelias venosas de cordão umbilical (ECV304), cultivadas em meio F12 com adição de 10 por cento de soro fetal bovino, foram plaqueadas e tratadas com concentrações clinicamente relevantes de Bevacizumab e Ranibizumab. As drogas foram administradas logo após risco realizado no meio da cultura (metodologia de scratch). Medidas lineares do espaço livre de proliferação celular foram realizadas 24, 48 e 72 horas após o momento da realização do risco. Todos os experimentos foram realizados em triplicata e a análise estatística foi feita pelo teste T-student. Resultados: O efeito inibitório foi observado em ambas as drogas, apenas nas concentrações 0,5 e 0,7 mg/ml. Na concentração 0,7 mg/ml, o Ranibizumab demonstrou efeito inibitório maior do que o Bevacizumab. Na mesma concentração, o Ranibizumab foi três vezes mais potente que o Bevacizumab. O efeito inibitório foi observado apenas nas primeiras 24 horas para ambas as drogas. Conclusão: O Ranibizumab demonstrou efeito maior quando comparado com o Bevacizumab, porém tal efeito está mais relacionado à diferença na razão molar das drogas do que relacionada com uma diferença real no efeito anti-proliferativo.

Purpose: To evaluate the comparative in-vitro antiangiogenic effect of Bevacizumab and Ranibizumab. Methods: Endothelial venous umbilical cells culture (ECV304) cultivated in F12 media with addition of 10 percent Fetal Bovine Serum, were plaqued and treated with clinically relevant concentrations of Bevacizumab and Ranibizumab just after the scratch done in the middle of the culture (scratch methodology). Measurements of the linear size of the area free of cell proliferation were done 24, 48 and 72 hours after the scratch day point. All the experiments were done in triplicate and statistical analysis were done with T-student test. Results: Inhibitory effect was observed just at the concentrations of 0.5 and 0.7 mg/ml in both drugs. At 0.7 mg/ml, Ranibizumab demonstrated a more potent proliferative inhibitory effect than Bevacizumab. At the same concentration, Ranibizumab was three times more potent than Ranibizumab. Inhibitory effect was observed just in the first 24 hours for both drugs. Conclusion: Ranibizumab demonstrates an increased effect when compared to Bevacizumab and this is related more to the different molar rate of each drug than related to a real better proliferative inhibitory effect.