Effect Of GM-CSF On Inducing Human Vascular Endothelial Cells To Form New Blood Vessels And The Role Of VEGF

IntroductionAS has been considering a kind of disease characterized by accumulation of lipid in the vascular wall during the past years. Recently researches have elucidated that inflammation plays a important role during AS pathological process, and finally contributes to unstable plaque development, even plaque disruption, and ACS. One of important characters of unstable plaque is angiogenesis within the plaque. Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, occurs in a variety of physiological and pathological settings, and shows extensive biological and clinical significances. The process of angiogenesis is very complex, which is controlled by both angiogenic factors and angiogen-ic inhibitors. Newly formed vessels provide ways for much more lipoprotein and contribute to plaque growth. What important is that a good many inflammatory cells influx through these newly formed blood vessels into plaque and produce a varieties of inflammatory factors, cytokines, growth factors, which aggressive inflammatory reactions within the plaque. Moreover, these newly formed blood vessels lack SMCs and mural cells themselves, which lead to plaque instability, even plaque disruption.Currently, researches about the influence brought by GM - CSF on angiogenesis are quit rare. Thus the experiment cultured HUVECs in vitro, and set up a stable angiogenesis culture system by Matrigel in order to discuss the influence brought by GM - CSF on inducing human vascular endothelial cells to form new blood vessels and the role of VEGF. Further discuss the relationship between GM - CSF, VEGF and AS progression.MethodsFirstly, HUVECs were cultured by modified Jaffe method and induced to develop tube structure. Thus set up a stable angiogenesis system and added the stimulating factors. Group one, divided into four groups, normal control (serum- free DMEM culture liquid) and rhGM - CSF stimulating groups in varying concentrations( 25>50^lOOng/ml) then acting 48h. Group two, divided into three groups, the same concentration rhGM -CSF(lOOng/ml) in varying stimulating time(l2N24^48h). Group three, divided into three groups, normal con-trol( serum - free DMEM culture liquid) ,rhGM - CSF( lOOng/ml) ,rhGM - CSF (lOOng/ml) + VEGF165 ( lOng/ml) then acting 24h. Then making varying groups fixed by 95% ice alcohol and calculating tube numbers in microscopic observations (xlOO). Finally analysis experiment results.ResultsThe experiment results had shown that, HUVECs can be induced to develop tube structure by Matrigel in vitro. After treatment with rhGM - CSF in varying concentrations(25N50N100ng/ml) , the number of tubes increased in a dose- dependent manner; and after treatment with rhGM - CSF of the same concentration but at varying time, with the time prolonged, the number of tubes increased as well in a time — dependent manner. rhGM - CSF( lOOng/ml) adding VEGF165(10ng/ml) compared with normal control (serum-free DMEM culture liquid) or rhGM - CSF( lOOng/ml) , the number of tubes increased evidently.DiscussionAS is a complex pathological process in a chronically progressive manner. Its clinic complication ACS - unstable angina pectoris, acute myocardial infarction, sudden cardiac death, are responsible for an exceedingly high number of deaths and disabilities over the past a century. Investigations have led to a betterunderstanding of how AS origin and progression. Currently, the role of inflammation in AS has been accepted widely. Interactions between lipoprotein, many kinds of cells, inflammatory factors, cytokines, growth factors go through the whole process, finally lead to unstable plaque, plaque disruption. And the formation of unstable plaque is tightly connected with newly formed blood vessels within the plaque. Angiogenesis is a process by which preexisting capillaries proliferate and form new capillary networks, including physiological condition ( embryonic development) and pathological condition ( AS, tumor growth, wound healing). This process include matrix degradation, endothelial cells migration , proliferation, tube formation. The mediator of angiogenesis can be classified by two kinds; angiogenic factors, such as VEGF, bFGF, IL and so on; angiogenic inhibitor, such as angiostatin, endostatin and so on. It was before o-ver one hundred years that angiogenesis within the plaque had been detected. When intima exceed a certain thickness, newly formed blood vessels originate from adventitial vasa vasorum going through media layer. With the development of lesion, the number, volume, shape of newly formed blood vessels are also changing. They provide ways for lipoprotein and contribute to plaque growth. What important is that a good many inflammatory cells influx through these newly formed vessels into plaque and produce a varieties of inflammatory factors, cytokines , growth factors, and MMPs expression up - regulation, which aggressive inflammatory reactions within the plaque. Moreover, these newly formed blood vessels lack SMCs and mural cells themselves, which lead to plaque instability, plaque disruption and a series of clinical acute complications.GM — CSF as a kind of inflammatory factor can be secreted by macrophage, SMCs, ECs within the plaque. The experiment cultured HUVECs in vitro, and set up a stable angiogenesis culture system by Matrigel in order to discuss the influence brought by GM - CSF on inducing human vascular endothelial cells to form new blood vessels and the role of VEGF. Results had showed that with the increase of GM - CSF concentrations and the adding time, the number of tubes were on rise. Moreover, the number of tubes also evidently increased after adding VEGF. Researches indicated that the effect of GM - CSF and VEGF on angiogenesis within the plaque can promote plaque instability, plaque disruption...