RESUMO
Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this, we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leukocytes to generate a same donor "vessel in a dish" bioassay. Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs), and leukocytes were obtained from four donors. Cells were treated in monoculture and cumulative coculture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumor necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines. Cocultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels. For the first time, we report a proof of concept study where autologous blood outgrowth "vascular" cells and leukocytes were studied alone and in coculture. This novel bioassay has usefulness in vascular biology research, patient phenotyping, drug testing, and tissue engineering.
Assuntos
Células Endoteliais/fisiologia , Leucócitos/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Bioensaio/métodos , Células Cultivadas , Técnicas de Cocultura/métodos , Citocinas/metabolismo , Descoberta de Drogas/métodos , Células Endoteliais/metabolismo , Humanos , Interleucina-6/metabolismo , Leucócitos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Engenharia Tecidual/métodos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm-inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine.
Assuntos
Produtos Biológicos/farmacologia , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Alemtuzumab , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Bioensaio/métodos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultura/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Reprodutibilidade dos Testes , Soro/química , Trastuzumab , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Haemophilus influenzae/fisiologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/microbiologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Animais , Células Endoteliais/citologia , Técnicas de Silenciamento de Genes , Infecções por Haemophilus/microbiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos Nus , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transplante de Células-Tronco , Receptor 4 Toll-Like/metabolismoRESUMO
Cyclooxygenase (COX) is required for prostanoid (e.g. prostaglandin PGE2) production. Constitutive COX-1 and inducible COX-2 are implicated in lung diseases, such as idiopathic pulmonary fibrosis (IPF). Using lung fibroblasts from humans and wild type, COX-1(-/-) and COX-2(-/-) mice, we investigated how COX activity modulates cell growth and inflammatory responses induced by activators of Toll-like receptors (TLRs) 1-8. In mouse tissue, PGE2 release from fresh lung was COX-1 driven, in lung in culture (24h) COX-1 and COX-2 driven, and from proliferating lung fibroblasts exclusively COX-2 driven. COX-2 limited proliferation in lung fibroblasts and both isoforms limited KC release induced by a range of TLR agonists. Less effect of COX was seen on TLR-induced IP-10 release. In human lung fibroblasts inhibition of COX with diclofenac was associated with increased release of IL-8 and IP-10. Our results may have implications for the treatment of IPF.
Assuntos
Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Citocinas/metabolismo , Fibroblastos/enzimologia , Proteínas de Membrana/genética , Receptores Toll-Like/agonistas , Animais , Proliferação de Células , Células Cultivadas , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Diclofenaco/farmacologia , Dinoprostona/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Imunidade Inata , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli I-C/farmacologia , Receptores Toll-Like/metabolismoRESUMO
Stem cell therapy and organ regeneration are therapeutic approaches that will, we suggest, become mainstream for the treatment of human disease. Endothelial cells, which line the luminal surface of every vessel in the body, are essential components in any organ regeneration programme. There are a number of potentially therapeutic endothelial cell types, including embryonic, adult progenitor and induced pluripotent stem cell-derived endothelial cells, as well as host vascular cells. The features (benefits as well as disadvantages) of each cell type that make them potentially useful in therapy are important to consider. The field of stem cell biology is well developed in terms of protocols for generating endothelium. However, where there is a distinct and urgent unmet need for knowledge concerning how the endothelial cells from these different sources function as endothelium and how susceptible they may be to inflammation and atherosclerosis. Furthermore, where stem cells have been used in clinical trials there is little commonality in protocols for deriving the cells (and thereby the specific phenotype of cells used), administering the cells, dosing the cells and/or in assessing efficacy attributed to the cells themselves. This review discusses these and other issues relating to stem cell-derived endothelial cells in cell therapy for cardiovascular disease.