Dataset Information

Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies.

ABSTRACT: Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to contract and promote the formation of new blood vessels that can supply the ischaemic region with nutrients and oxygen. This study focused on the genetic modification of human skeletal muscle cells (SkMCs). We chose myoblast cells due to their close biological resemblance to cardiomyocytes and the placental growth factor (PlGF) gene due to its pro-angiogenic potential. In our in vitro studies, we transfected SkMCs with the PlGF gene using electroporation, which has previously been proven to be efficient and generate robust overexpression of the PlGF gene and elevate PlGF protein secretion. Moreover, the functionality of the secreted pro-angiogenic proteins was confirmed using an in vitro capillary development assay. We have also examined the influence of PlGF overexpression on VEGF-A and VEGF-B, which are well-known factors described in the literature as the most potent activators of blood vessel formation. We were able to confirm the overexpression of VEGF-A in myoblasts transfected with the PlGF gene. The results obtained in this study were further verified in an animal model. These data were able to confirm the potential therapeutic effects of the applied treatments.

SUBMITTER: Zimna A

PROVIDER: S-EPMC5851700 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies.

Zimna Agnieszka A Wiernicki Bartosz B Kolanowski Tomasz T Rozwadowska Natalia N Malcher Agnieszka A Labedz Wojciech W Trzeciak Tomasz T Chojnacka Katarzyna K Bednarek-Rajewska Katarzyna K Majewski Przemyslaw P Kurpisz Maciej M

Archivum immunologiae et therapiae experimentalis 20170926 2

Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to c ...[more]

PMID: 28951939

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Project description:Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores, coupled with their desiccation resistance, makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes, one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of GMO spores by utilizing the concept of a thymineless death, a phenomenon first reported 6 decades ago. Using Bacillus subtilis, we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB, using a two-step process. Insertion is made first at thyA and then at thyB whereby resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine), and in its absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes.IMPORTANCE Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health, including their use as heat-stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores released into the environment where they could survive in a dormant form indefinitely. We describe a solution whereby, following genetic manipulation, the bacterium is rendered dependent on thymine. As a consequence, spores if released would produce bacteria unable to survive, and they would exhibit a thymineless death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a critical problem for containing spores of GMOs in the environment.

Project description:BACKGROUND: Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance. METHODS: The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated. RESULTS: The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli. CONCLUSIONS: We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.

Dataset Information

Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies.

Publications

Biological and Pro-Angiogenic Properties of Genetically Modified Human Primary Myoblasts Overexpressing Placental Growth Factor in In Vitro and In Vivo Studies.

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