Effects Of Carbon Nanotubes On Vascular Endothelial Cells Damage And Its Mechnism

The most attractive properties of nanomaterials for medical and technologic applications, including their small size, large surface area, and high reactivity, are also the main factors for their potential toxicity. Based on their inhalation studies with ambient ultrafine particles, it has been predicted that nanosized particles will have higher pulmonary deposition and biological activity compared with larger particles. Thus, some nanosized materials may induce not only damage at the deposition site but also distant responses as a result of their translocation and/or reactivity through the body. In this respect, epidemiologic and experimental studies have suggested an relation between respiratory exposure to ambient ultrafine particles ( including particles with a diameter <100nm ) and the progression of cardiovascular disease. Engineered carbon nanomaterials, including carbon nanotubes, have elicited a great deal of interest recently because of their unique electronic and mechnical properties. Carbon nanotubes(CNT ) have a diameter ranging from 0.7 to 1.5nm, with lengths >1μm. Initial toxicologic studies demonstrated that intratracheal or pharyngeal instillation of CNTs suspension in mice caused a persistent accumulation of carbon nanotube aggregates in the lung, followed by the rapid formation of pulmonary granulomatous and fibrobic tissues at the site. The unique physical characteristic and the pulmonary toxicity of CNTs raised concerns that respiratory exposure to these materials might be associated with systemic toxicities. In the present studies, we demonstrate that lung instillation of CNTs is associated with a dose-dependent increase in oxidative modifications, which are considered to play a role in atherogenesis, we further evaluated the effects of CNTs respiratory exposure on rat.Since endothelium is the first layer of vascular, endothelial cells would be the target to be injured firstly. The cell adhesion molecules including ICAM-1 and VCAM-1 are important for binding of leukocytes to the endothelial cells and in the infiltration of inflammatory cells into tissues. Nuclear factor-kB (NF-kB) has been implicated in the transcriptional activation of the genes encoding CAMs. Rapid phosphorylation and degradation of IkBαallows NF-κB to translocate into the nucleus and to regulate transcription of the target genes. As very little is known in regard to the toxicology and the underlying mechanism involved in explaining the phenomena of carbon nanotubes (CNTs) exposure, we carried out a detailed study on their effects of the expression of cell adhesion molecules on rat aortic endothelial cells ( RAECs). We demonstrate here that CNTs induced adhesion of neutrophils to endothelial monolayer by promoting the expression of ICAM-1, VCAM-1. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB/P65 activation in CNTs treated RAECs. As oxidative stress is known to regulate the activation of NF-κB, we tested GSH content and LDH activity. We found that CNTs induced oxidative stress and调the expression of ICAM-1 and VCAM-1. The nucleus translocation of NF-κB/P65 after CNTs exposure could be inhibited by N-acetylcysteine which indicated that the high expression of ICAM-1 and VCAM-1 mediated by oxidative stress in rat aortic endothelial cells might play an important inflammation role in CNTs-induced vascular endothelium damage. After establishing RAECs damage model, we measured the expression of ICAM-1 and VCAM-1 in cultured RAECs stimulated with different dose of CNTs for different time exposure and detected NF-κB signaling pathways.Method :we evaluated the toxic effects of intratracheal instillation of CNTs on rats and CNTs exposure on RAECs. The related indexes including oxidative damage, inflammatory factor, adhesive molecular and signal molecular were detected by biochemical assays, immunological histochemistry, RT-PCR and Western blot. Data analyzed by using SPSS1 .0 software.Result:1. Animal test:Rats were intratracheally instilled with 0, 0.7, or 3.5 mg of carbon nanotubes, carbon black as negative control, or quartz as positive control. The rats were euthanized on 7 d or 30 d after the single treatment per day for study of histopathological changes in some organs and expression of sICAM-1 and sVCAM-1in rats'blood.1)Histopathological examination:Histopathological evaluation of lung tissues revealed that lung in rats exposed to CNTs and quartz particles produced a dose-dependent lung inflammatory response characterized by neutrophils infiltration and foamy (lipid-containing) alveolar macrophage accumulation. In addition, lung tissue thickening as a prelude to the development of fibrosis was evident and progressive. Histopathological evaluation of aortic vascular tissue revealed that CNTs exposure fior 30d resulted in a protuberance on inner membrane of aortic vascular, but CB and SiO2 groups had no this kind phenomenon.2)Blood routine examination and blood biochemistry assay:The results of blood routine examination showed that only the contents of PLT,WBC and ?b value were different between NS control group and CNTs,SiO2 experimental groups, especially in CNTs experimental group these index showed the highest value. The results of LDH activity, CK,ALT,AST and sEPCR contents increased in a dose dependant manner and time dependant manner under treatment with all particle types especially the highest release induced by SiO2 at a particle concentration of 17.5 mg/kg. However, maximal LDH release induced by CNTs treatments was noted to be lower than that observed with SiO2 (positive control) possibly due to high particle concentrations interfering with the assay.3)Oxidative stress and formation of reactive oxygen species:A significant depletion of GSH was observed at high dose of CNT exposure compared with control group, and was also time- dependent. But the content of O2ˉ·increased with the time and dose of CB,CNTs and SiO2 exposure, and a significant increase in CNTs and SiO2 exposure compared with control group. Overall, the results demonstrated a significant depletion of GSH and increase in oxidative damage levels occurred in CNTs exposed rats.4)Inflammatory response (TNF-αand IL-1β):Our results showed that CNTs exposure elevated the expression of TNF-αand IL-βin dose and time dependent manner, although there was only weak expression in low dose group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, and the expression level of these two inflammatory factors in rats exposed to CNTs exposure was greater than that of CB and SiO2 at the same dose and the same time.5)sICAM-1 and sVCAM-1 expression of blood:CNTs increased monocyte adhesion to cells dose-dependently and time-dependently. Meanwhile, CNTs exposure elevated the expression of sICAM-1 and sVCAM-1 in dose and time dependent manner, although there was only weak expression in low dose group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, and the expression level of these two adhesion molecular in rats exposed to CNTs was greater than that of CB and SiO2 at the same dose and the same time.6)Immunohistochemistry of ICAM-1 and VCAM-1 on aortic vascular after CNTexposure:The results of immunohistochemistry showed that ICAM-1 and VCAM-1 only expressed infirmly in endothelial cells of NS control group , but VCAM-1 expressed positively in endothelial cells and protuberance of CNTs experiment group, which were located in cytoplasm, cytomembrane and intercells; ICAM-1 expressed firmly in protuberance including endothelial cells even smooth muscle cells of midmembrane.2.Cell test:RAECs were exposed to CNTs suspension at different dose and for different time.1)In vitro cell oxidative stress: A significant depletion of GSH was observed at dose 50-200μg·mL-1 of CNTs compared with that in the control group. Overall, the results demonstrated a significant depletion of GSH levels occurred in RAECs exposed to CNTs. The results of LDH leakage and exhibited a significant cytotoxicity at CNTs concentrations of 12.5-100μg·mL-1. There was a statistically significant difference between different dose of CNTs, whereas the highest toxicity was at 100μg·mL-1 but not 200μg·mL-1.The detailed mechanism is not clear, which needs more work to do.2)CNTs localization and ultrastructure observation: After exposure to CNTs, there were clear changes in the ultramicroscopic features of RAECs. CNTs frequently lodged inside cytoplasm and mitochondria. RAECs incubated with CNTs showed extensive disruption of mitochondrial cristae, resulting in a vacuolar cellular appearance and microvillus. These changes were time dependent, with fewer particles collecting inside mitochondria during shorter incubations. Electron microscopy showed considerable mitochondrial damage by CNTs, resulting in the formation of concentric structures, known as myelin figures. 3)Cell viability:The MTT assay showed CNTs was toxic to RAECs and the cytotoxicity increased with CNTs doses and exposure time. From these results, we could see that most mitochondria were damaged characteristically by swelling with architectural disruption which was consistent with the qualitative analysis.4)SEM image of adhesion between RAEC and PMBC after CNTs exposure:Under the SEM, RAECs grew as a monolayer, adhering to culture bottle wall like creeping. Cells'border was not tidy, surface not smooth and had silk-like salience. RAECs could connect each other by these saliences. PBMC were like ball and their surfaces were not smooth which looked like microvillus. RAECs adhered to PMBCs through these saliences under SEM. PMBCs could also adhere to RAECs by microvillus on their surfaces. Secondary-tethering phenomenon could be observed: connection between PMBCs which jointed to the surfaces of RAECs or backstop adhered to the surface of RAECs provided a point to adhere to other PBMCs which led to strengthen and enlarge adhesion between RAECs and PMBCs. The degree of adhesion increased with the doses of CNTs.5)Expression of ICAM-1 and VCAM-1 protein and mRNA:CNTs exposure elevated the expressions of ICAM-1 and VCAM-1 in dose and time dependent manner.By one way analysis of variance,the expression level of ICAM-1 and VCAM-1 in vascular cellular endothelium in high dose groups (25-200ug·mL-1) was statistical higher than that in the control group.And in the case of the same dose treatment, the longer the CNTs exposure, the higher expression level of them occurred, but the difference of expression level between exposure for 12h and 24h was greater than that for 24h and 48h. In agreement with the protein data, noticeable changes were detected in the steady-state mRNA levels of both ICAM-1 and VCAM-1.6) CNTs exposure induced activation of NF-κB, IKKαand the degradation of IκBα:Comparison of the levels of NF-κB in the nuclear fraction indicated that the level of NF-κB in the nuclear fraction was higher in CNTs exposure cells than that in the control cells. Western blotting and densitometric analysis of the density of bands relative toβ-actin indicated that CNTs exposure resulted in markedly greater activation of NF-κB and its translocation to the nucleus than that in control RAECs. However, the CNTs induced activation and translocation of NF-κB to the nucleus in the RAECs was greater than that in the control RAECs; Western blot analysis and subsequent measurement of density of bands relative toβ-actin indicated that activation level of IKKαwas higher in CNTs exposure RAECs than that in the control RAECs. Greater degradation of IκBαwas noted in CNTs exposure RAECs as compared to the control RAECs.7)Effects of N- acetylcystein on nuclear translocated NF-κB/p65 induced by CNTs in RAECs:The immunoanalysis of effects of arecholine on nuclear translocated NF-κB/p65 induced by CNTs in RAECs showed that CNTs exposure increased the nuclear translocated NF-κB/p65 compared with that in the control group, however pretreating the cells with N-acetylcystein before CNTs exposure, the nuclear translocated NF-κB/p65 decreased but was still higher than that in the control group. It suggested that N-acetylcysteine could inhibit CNTs to induce NF-κB/p65 expression on rat aortic endothelial cells. ROS production and oxidative stress were probably some of the mechanisms.Conclusion:1. An important consideration is that oxidative stress, and especially depletion of reduced GSH and O2ˉ·production, the leakage of IL-1βand TNF-αincreasing with dose and time of CNTs exposure, which is the same as the results of sICAM-1 and sVCAM-1. These results show that, for the test conditions described here and on an equal-weight basis, carbon nanotubes can pass through lung-blood barrier and reach the blood, they are much more toxic than carbon black and can be more toxic than quartz, which is considered a serious occupational health hazard in chronic inhalation exposures. As markers of endothelial cells damage, the three indicators of experimental animals especially CNT group are higher than those of control group. In this study, we have sought to bring these findings to suggest pathobiologic processes whereby nanaoparticles, especially CNTs, had effects on the cardiovascular system. Pathologic end points relevant to plaque rupture, endothelial erosion, hemostasis, and coagulation should be used in toxicologic studies.2. The results of cell tests showed that CNTs exposure can induce RAECs damage which including ultrastructure of RAECs changed and cell viability decreased, especially the expression of ICAM-1 and VCAM-1 mRNA and protein are higher than that in control RAECs.3. We demonstrate here that CNTs induce adhesion of neutrophils to endothelial monolayer by promoting the expression of ICAM-1, VCAM-1. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB/P65 activation in CNTs treated RAECs. As oxidative stress is known to regulate the activation of NF-κB, we tested GSH content and LDH activity. We found that CNTs induced oxidative stress and the expression of ICAM-1 and VCAM-1. The nucleus translocation of NF-κB/P65 after CNTs exposure can be inhibited by N-acetylcysteine, which indicate that the high expression of ICAM-1 and VCAM-1 mediated by oxidative stress in rat aortic endothelial cells may play an important inflammation role in CNT-induced vascular endothelium damage.