RESUMO
Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.
Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Hemorreologia , Estresse Mecânico , Animais , Bovinos , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Endotelinas/biossíntese , Endotelinas/genética , Epoprostenol/biossíntese , Epoprostenol/genética , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Mecanorreceptores/fisiologia , Membranas Artificiais , Óxido Nítrico/biossíntese , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , VácuoRESUMO
The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hb's inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.
Assuntos
Endotelinas/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Hemoglobinas/farmacologia , Animais , Bovinos , Células Cultivadas , Endotelinas/biossíntese , Endotelinas/imunologia , Endotélio Vascular/lesões , Hemoglobinas/química , Hemoglobinas/imunologia , Hemoglobinas/metabolismo , Humanos , Imunoquímica , Peso Molecular , Oxirredução , Polímeros/química , Polímeros/farmacologiaRESUMO
OBJECTIVE: To investigate the effects of nitric oxide (NO), endothelin (ET), substance P (SP) and calcitonin gene-related peptide (CGRP) immunocompetence substances and their relationship to chronic periodontitis. METHODS: Immunohistochemical aod histochemical staining methods were used to detect the expression of the NO synthase (NOS), ET, SP and CGRP levels in 20 patients with chronic periodontitis and 20 healthy subjects as control. RESULTS: Quantitative analysis by Quantimat 970 showed that NOS and ET in periodontitis tissue increased significantly (P < 0.01), particularly the content of ET in comparison with healthy subjects. The intergroup expression of SP and CGRP showed no remarkable changes. CONCLUSION: Our results demonstrate that the level of NOS and ET were significantly increased in periodontic tissue, which may diminish the blood supply and influence the periodontal tissue causing tissue damage. Our study suggests that immunocompetence substances NO and ET are closely associated with periodontitis and may play an important role in the disease.
Assuntos
Gengiva/química , Óxido Nítrico/biossíntese , Periodontite/metabolismo , Biossíntese de Proteínas , Adulto , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Doença Crônica , Endotelinas/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Masculino , Periodontite/patologia , Substância P/biossínteseRESUMO
Polymeric structures of a polylactide-polycaprolactone blend were micro-fabricated using the Pressure Assisted Microsyringe (PAM) system. Human umbilical vein endothelial cells were cultured on the scaffolds, and apoptosis, cell adhesion, proliferation and metabolism were evaluated. In addition, more specific indicators of endothelial cell function, namely nitric oxide and endothelin production, were also assessed. Thin films of the blend, as well as gelatine-coated glass slides (as controls) were used. The results show that as far as adhesion, apoptosis and metabolism are concerned, the scaffolds do not interfere with cell function compared with gelatin controls. However, the nitric oxide/endothelin ratio was higher than that observed on the gelatin films, suggesting that the scaffolds could be used for engineering small diameter blood vessels without risk of occlusion.
Assuntos
Sistema Cardiovascular/citologia , Células Endoteliais/citologia , Polímeros/química , Engenharia Tecidual/métodos , Adesão Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Endotelinas/biossíntese , Gelatina , Humanos , Interações Hidrofóbicas e Hidrofílicas , Óxidos de Nitrogênio/metabolismo , Silanos/química , Engenharia Tecidual/instrumentação , Cordão Umbilical/citologiaRESUMO
A method for the expression of recombinant DNA products in mammalian cells based on in vitro amplification of gene units is described. Gene cassettes containing either a selectable marker or the gene of interest are mixed at different molar ratios, and linear polymers are formed using forced head-to-tail ligation. After introduction of the polymers into mammalian cells, transformants with various amounts of the amplified gene unit are obtained. For a first characterization of the system, the gene coding for chloramphenicol acetyltransferase (CAT) has been used to produce polymers containing a single neomycin-resistance gene ligated to different numbers of CAT gene units and used for transfection into HeLa cells. All isolated G418 (gentamycin)-resistant cell transformants which received in vitro amplified DNA were found to express high levels of CAT activity in a stable manner. Southern-blot analysis of individual clones revealed multiple copies of the gene integrated head-to-tail in the genome. This system allowed us to express the gene coding for human prepro-endothelin-1 in A617 human vascular-smooth-muscle cells. The recombinant protein was shown to be correctly processed and biologically active endothelin-1.