Effect Of Multi-walled Carbon Nanotubes On Endothelial Dysfunction

ObjectivesMulti-walled carbon nanotubes(MWCNTs)are nanofiber materials with excellent electrical,thermal and mechanical properties,and have been widely used in many fields such as batteries,ships,and sports equipments.In addition,the unique hollow structure of MWCNTs makes it possible for the available cavity to contain bio-specific molecules and drugs.The outstanding biocompatibility guarantees the combinition with proteins,nucleic acids,lipids and other biomolecules.These characteristics of MWCNTs determine the probable applications such as drug carriers in the field of biomedicine.Recently,researches regarding on the adverse effects of MWCNTs mostly focused on the respiratory toxicity caused by occupational exposure,while those on other organs or tissues were few.However,the cardiovascular system was the organ most susceptible to MWCNTs when exposed directly as drug carrier.Therefore,it was of paramount significance to clarify the toxic effects of MWCNTs on the cardiovascular system and to explore related mechanism to provide scientific bases for the safety evaluation and risk assessment of MWCNTs products.Providing above,this study combined experiments in vivo and in vitro to clarify the endothelium dysfunction caused by MWCNTs,and further explored the role of VEGF in this damage effect.Methods1.Test substance: MWCNTs,length 10-20 ?m,diameter 10-50 nm and >50 nm.2.Experimental subjects: human umbilical vein endothelial cells(HUVECs)and C57BL/6J mice.3.The amount of reagents and the way of exposure:(1)HUVECs: MWCNTs was given at doses of 0.1,1,10,100 ?g/m L.(2)C57BL/6J mice: the experimental group was given 2 mg/kg of MWCNTs via the tail vein,or intraperitoneal injection of Matrigel 0.5 m L containing MWCNTs 10 ?g/m L.4.Experimental methods and content(1)Transmission electron microscopy(TEM)was applied to figure out whether MWCNTs could enter HUVECs.(2)CCK-8,LDH,and Calcein-PI/AM methods were adopted to investigate the effects on cell viability.Tubule formation experiment and cell scratch test were conducted to investigate the injuries on capacities concerning regeneration and migration.(3)HE staining of lungs and aorta diagrams were analyzed to investigate the adverse effect of MWCNTs on the small and large blood vessels of mice.By the Matrigel Plug model,influences of MWCNTs on the angiogenesis ability of mice were explored.(4)Protein chip technology was used to manifest the effect of MWCNTs on the expression of vascular function-related proteins in HUVECs culture supernatant.VEGF levels in HUVECs culture supernatant and mouse serum were detected by ELISA,and downstream proteins,AKT and eNOS,were quantified by Western-Blot,with which the molecular changes indicated.(5)The addition of recombinant VEGF to the MWCNTs treatment group accomplished the observation on changes of HUVECs tubule forming ability and of vascular regeneration ability of mice,meanwhile alterations of the levels of AKT and eNOS were detected,to investigate the role of VEGF in MWCNTs-induced endothelium dysfunction.ResultsThe results were as follows:1.TEM showed the presence of MWCNTs within HUVECs.2.CCK-8 and Calcein-PI/AM showed that the cell viability of the experimental group was lower than that of the control group,while the LDH method showed similar changes when the concentration of MWCNTs reached 100 ?g/m L.Cell function experiments showed the length of tubule formed was inferior to that of the control group,the wound healing rate lower than that of the control group.The above differences are statistically significant.3.After tail vein administration,MWNCTs particles were deposited in the lungs of the mice,where thickening of the lung interval and granuloma formation were found,whereas no obvious pathological changes were seen in the aorta.The angiogenesis experiment showed relatively inferiority of neovascularization in the experimental group,the difference of which was statistically significant.4.The protein chip results showed the reduction on the expression of vascular function related proteins in the experimental group.ELISA results showed that the VEGF levels both in the HUVECs supernatant and in mice serum of the experimental group were lower than those of the control group.Western-Blot results showed that the expression levels of AKT and eNOS in the experimental group were inferior to those in the control group.5.After adding recombinant VEGF,the length of tubule and the number of neovascularization in the experimental group increased significantly.ConclusionsThrough the above experiments,the conclusions were as follows:1.MWCNTs could affect cell viability,reduce the tubule forming ability and wound healing ability of HUVECs,and weaken the vascular regeneration ability of mice,suggesting that MWCNTs can cause endothelial dysfunction.2.MWCNTs could down-regulate HUVECs VEGF expression,mouse serum VEGF levels,and downstream AKT and eNOS expression.The addition of recombinant VEGF relieved the functional damage caused by MWCNTs,increased the expression of AKT and eNOS,suggesting MWCNTs-induced endothelial dysfunction was achieved by inhibiting VEGF/AKT/eNOS3.MWCNTs with diameter 10-50 nm and over 50 nm caused similar endothelial function damage,suggesting that in the MWCNTs investigated,the diameter factor generated no significant effects on vascular endothelial toxicity.