The Effects Of Granulocyte Colony-stimulating Factor (G-CSF) On Chronic Obliteration In The Artery System

In recent years,a large number of animal experiments and clinical studies confirmed that stem cell transplantation can improve cardiac function after myocardial infarction,and prevent ventricular remodeling,thereby improve the prognosis.Stem cells are a kind of undifferentiated cells at early stage which have multi-directional differentiation potential and self-renewal,and can differentiate into different functional cells under certain conditions to form various of tissues and organs,termed as "plasticity".Granulocyte colony-stimulating factor(G-CSF) is the cytokine that is widely used in patients with radiotherapy and chemotherapy to mobilize hematopoietic stem progenitor cells for restoration support.Recent studies have shown that G-CSF can mobilize bone marrow stem cells into circulation, migrate to the site of myocardial infarction to prompt myocardial regeneration and angiogenesis,and improve heart function after myocardial infarction.As a non-invasive treatment,there is huge potential for the application of G-CSF in the treatment of myocardial infarction.Studies in the chronic ischemic heart disease also show the same view.However,most of the studies of G-CSF focused on its effect on heart,its impact on vessels especially chronic obstructive artery diseases related to ischemic heart diseases such as thrombosis and atherosclerosis,is rarely reported. Furthermore,G-CSF motivation can influence blood system by inducing leukocytosis, resulting in hypercoagulation.It is still not clear whether G-CSF can induce the deterioration of arterial occlusive disease.In short,the impact of G-CSF on local arterial vascular system and chronic obstructive can not be determined in domestic and foreign academics.Many recent researches have shown that,as a sort of the stem cells,endothelial progenitor cells(EPCs) exist in bone marrow,peripheral blood and cord blood.The EPCs can be recruited to the local vascular injury,differentiated into mature endothelial cells,and involved in angiogenesis and repair by stimulated with injury signals.There are various methods to stimulate EPCs in peripheral blood including exercise,drugs,etc.The drugs which can increase EPCs in adult peripheral blood include stem cell factor,granulocyte colony-stimulating factor,statins,estrogen and so on confirmed by the current researches.As an exogenous stem cell mobilization agent,G-CSF can stimulate EPCs from bone marrow into the peripheral blood and accelerate re-endothelialization of the injury vascular.With the combination of well-known adverse reactions,we infer that G-CSF can be bound to have repair effects and even side effects on the local blood vessels,including chronic obstructive pulmonary arterial lesions and arterial system itself.As a vascular injury,chronic arterial occlusive diseases are a series of common cardiovascular diseases threatening human health.The diseases are not only the altered shape and structure of blood vessels,but more importantly,the changes of functions.There is minimal study about the role of G-CSF on it especially.So,this study expounded two aspects of the problem systematically from the morphology and function.The research was divided into four parts:firstly,a new type of chronic arterial occlusive disease model was set up.Secondly,combined with the relatively mature rabbit atherosclerosis model,we analized the effects of G-CSF on the local endarterium with chronic obliteration in artery.Thirdly,we observed the effects of G-CSF on different types of arterial lesions as well as chronic obstructive arterial system was done.Finally,we studied G-CSF on platelet activated function to verify whether it is involved in chronic arterial obstruction which can make a preliminary summary of the whole study and explore a new direction for the next step.Partâ… Establishment and imaging evaluation of the chronic carotid artery obstructive model of rabbitObjective:To set up a new type of rabbit chronic carotid obstructive arteries model for further studies of the effect of G-CSF on chronic obstructive arterial lesions.Methods:Eighteen New Zealand white healthy rabbits were randomly divided into the sham-operated group,4-week group and 8-week group,each including 6 rabbits. The artery narrowing rings were implanted in the right carotid to prepare the chronic artery occlusion model.Carotid artery ultrasound and magnetic resonance vascular imaging study were used for the model evaluating.The animals were sacrificed after 4 weeks(4-weeks group) or 8 weeks(8-weeks group).The proximal and distal vessel tissues of the chronic obstructive narrowing rings were separated and performed the histopathological examination,then the structure and function changes of partial endothelial cells in obstructive region were assessed.Results:Seventeen rabbits survived and 11 were successfully set up chronic carotid obstructive artery models in a total of 18 rabbits.Carotid artery obstruction was observed gradually after implanting artery constrictor lasting for 4 weeks.Carotid artery ultrasound follow-up showed that vascular diameter from(2.00±0.1)cm preoperatively decreased to(1.33±0.23)cm,and the rate of stenosis changed to(48±7)%.Vascular diameter was significantly decreased to(0.48±0.06)cm while the rate of stenosis reached(95±3)% at 8weeks,compared with the control group and 4-weeks group(P＜0.05),while blood flow velocity and resistance index also changed significantly.Rabbit carotid artery model of magnetic resonance showed that the former is clearly visible,bilateral symmetrical,and vascular diameter was(2.02±0.15)cm,but the stenosis significantly reached(1.56±0.18)cm on the constrictor site of the right common carotid artery at 4 weeks,meanwhile the artery was almost completely blocked and had only traces of residual thin line at 8 weeks,the difference has statistical significance compared with the sham-operated group(P＜0.05).Pathological examination of related vessel model revealed that the intima continued thickening,extracellular matrix increased,resulting in severe luminal stenosis or occlusion.Scan electron microscope confirmed endothelial cell apoptosis after chronic obstruction,and matrix surface granular material increased.The ET-1 gene expression was up-regulated and the eNOS gene expression was down-regulated in distal vascular of chronic obstructive artery by Real-time PCR,meanwhile the compensatory changes in contralateral arteries were observed.Conclusions:Chronic arterial occlusive disease animal model can be successful set up 4 weeks after implanting artery constrictor.It is more significant after 8 weeks,and the endothelial morphology and function changes of corresponding paragraph of vessel were observed.Partâ…¡The effects of granulocyte colony-stimulating factor(G-CSF) on the local vascular paragraph of chronic obstructive arterial lesionsObjective:To observe the partial functions and matrix changes of blood vessel during application of G-CSF on chronic artery occlusion animal models,and to explore the impacts of G-CSF on the local vessel of the chronic obstructive arterial disease during treatment of cardiovascular diseases,then evaluate its safety,so as to provide a new theoretical basis for clinical practice.Methods:Thirty-six New Zealand white rabbits were first randomly divided into high-fat feeding group and ordinary feeding group,and then each subdivided into 3 subgroups named separately sham-operated group,chronic arterial occlusive model group and chronic arterial occlusive model add G-CSF-intervention group,each group had 6 rabbits.The atherosclerosis model was set up by using high-fat feeding;the method of G-CSF intervention was 15ug/kg/d,subcutaneous injection for 5 days,while using Giemsa staining and white blood cell count to confirm the mobilization results.The blood routin and lipid biochemical detection were done before and after model was made, then molecular(PCR and WB)experiments and pathological analysis were done based on the local carotid artery of the chronic obstructive and the corresponding parts after 2 months.Bilateral carotid artery ultrasound follow-up check was given at 3,5,7weeks after model was set up;collagen staining,MMP-9 and TIMP-2 immunohistochemical check and WB were detected to study the structure changes in vascular matrix.The form and function changes of the corresponding vascular endothelial cells were assessed by using scanning electron microscope,Real-time PCR detection of eNOS and ET-1mRNA expression.Results:Twenty-four animal models for chronic obstructive common carotid arteries were successfully established. Blood lipid items like TC and LDL were significantly elevated in the three high fat feeding groups 2 months after feeding.AI(Arteriosclerosis Index) was elevated from (1.21±0.16) to(2.70±0.74)(P＜0.05).Granulocytes were significantly increased by Giemsa staining after G-CSF motivation,correlated with the increase of WBC counting.Shrinked internal diameter,slowed bloodstream,and increased resistance index of the proximal end of common carotid artery was observed using carotid artery ultrasound 3 weeks after operation(P＜0.05).Stenosis appeared more dramatic in high fat feeding rabbits after the application of G-CSF[from(53.24±0.7)%to (59.51±0.5)%(P＜0.05)].Vessels were consecutively clogged until completely occlusion 5 weeks later,with a more dramatic change in arteriosclerosis vessels confirmed by the ultrasound.It took about 7 weeks for complete occlusion of the vessel in normal feeding rabbits,and it was 2 weeks shorter in the G-CSF group. Tunica intima proliferation and endothelium reductus growth were observed by HE staining in the G-CSF group.Arteriosclerosis in the common carotid artery of rabbits presented as decreased muscle fiber,increased collagen and amyloidosis tissues by collagen staining in the high fat feeding group,which was more obvious in the proximal end of vessel during chronic obstruction,and the change was more dramatic in the G-CSF group.Like those in arteriosclerosis,expression of MMP-9 and TIMP-2 were up regulated in the G-CSF group confirmed by Western blot,and exacerbated clogging.Increased eNOS expression and decreased ET-1 expression in the distal end of the vessel and a compensation of the opposite side were observed using Real-time PCR in the G-CSF group(P＜0.05).Endothelium hyperplasy in the proximal end of chronic obstructive vessel was observed in the G-CSF group using scanning electron microscope,the alternation was limited in turbulent flow.Conclusions:G-CSF has the ability to improve endothelial function through inducing endothelialization. However,like arteriosclerosis induced by high fat feeding,chronic obstructive artery lesions are likely to be worsened by G-CSF due to deterioration of arteriosclerosis and gelatinous fiber hyperplasia induced by increased matrix metalloproteinase in vessels.Partâ…¢The impact of G-CSF on Chronic Obstructive Artery SystemObjective:To systematically investigate the impact of G-CSF on various types of artery endothelial cells in arteriosclerosis and analyze its systematic impact on chronic obstructive artery systems like arteriosclerosis from functional and morphological aspects,with the hope of providing a guidance for a better application and prevention of adverse effects in clinical practice.Methods:Thirty healthy experimental New Zealand white rabbits were randomly divided into the control group(n=6),the normal feeding group(n=6),normal feeding +G-CSF group(n=8),the high fat feeding group (n=8),the high fat feeding +G-CSF group(n=8).G-CSF was injected subcutaneously at a dose of 15ug/kg diluted to 0.5mL with normal sodium for a consecutive 5 days,4 times in all once every other week.Blood sampling was collected once a week.NO concentration was assessed by nitrate reductase method.Blood lipid concentration was assessed by automatic blood biochemistry analyzer.Adipose fat plague in thoracic aorta was analyzed by Sudanâ…¢adipose staining 10 weeks later.Intima thickness of common carotid artery,femoral artery,and thoracic aorta were analyzed using HE staining.Thoracic aorta Matrix was analyzed by collagen staining and immunohistochemical test.The mRNA expression of MMP-9,TIMP-2,eNOS and ET-1 in common carotid artery,femoral artery,and thoracic aorta were analyzed using RT-PCR.Protein expressions of MMP-9 and TIMP-2 in various arteries were analyzed using Western-Blot.Surface appearance of various artery endothelial cells was observed using scanning electron microscope.Altered ultrastructure in endothelial cells in thoracic aorta like mitochondria was analyzed using transmission electron microscope.Results:Eight rabbits died during experiment.There finally survived 5,6,5,6 animals in the four groups,respectively.Blood lipid dynamic test found an elevated blood lioid like TC and LDL in the high fat feeding group,reaching peak 6 weeks later(P＜0.05),stable 8 weeks later.The application of G-CSF shifted forward the peak of blood lipid such as TC and LDL by 2 weeks(compared with the control group,P＜0.05),presenting double hump,with no influence on TG.NO monitoring showed sharp elevation of serum NO by G-CSF(compared with the control group 2 weeks later,P＜0.05).The high fat feeding group showed a slow elevation in 4 weeks(P＜0.05),and the G-CSF intervention group showed the most significant change(P＜0.01).Adipose fat plague in thoracic aorta was observed by Sudanâ…¢adipose staining in the high fat feeding group.G-CSF expanded the plague from(57.40±1.68)%to(91.18±2.27)%(P＜0.05).An elevation of intima thickness was found in the normal feeding group from(8.77±0.89)%to(11.10±1.50)%using HE staining,while a change from(14.97±1.87)%to(25.83±1.88)%in the high fat feeding group was found(P＜0.05).Collagen staining in thoracic aorta suggested that G-CSF induced production of matrix.Increased positive rate of MMP-9 and TIMP-2 were confirmed by immunohistochemical test[(1.08±0.06)%,(26.31±1.08)%, (27.90±1.07)%,(45.51±1.60)%,(4.07±1.01)%,(36.08±1.68)%,(71.36±3.01)%, (67.26±2.54)%,respectively in the four groups],RT-PCR confirmed a similar change of MMP-9 and TIMP-2 mRNA expression.Altered eNOS and ET-1 mRNA expression suggested the severity of ET-1,an index reflexing the impaired endothelial function,was that thoracic aorta＞common carotid artery＞femoral artery,and the expression of eNOS gene,reflexing a protective effect,changed the opposite in the model of arteriosclerosis induced by high fat feeding.G-CSF induced an up-regulation of the expression of eNOS gene in normal feeding group and a down-regulation in the high fat feeding group,meanwhile eNOS expression changed the opposite.This was more dramatic in thoracic aorta and less significant in middle and micro vessels like common carotid artery and femoral artery.The electron microscope found increased apoptosis of EC by high fat diet and increased EC by G-CSF.The latter showed the most significant in normal arteries and less in the surface of arteries in arteriosclerosis.Ultrastructure of endothelial cells like mitochondria was dramatically changed by high fat feeding and G-CSF using transmission electron microscope,which changed more significantly when they were both there.Conclusions:G-CSF deteriorated arteriosclerosis presenting as expensed plaque,thickened tunica intima mainly through impacting TC and LDL,not TG, possibly related to its impact on matrix.Model of arteriosclerosis was successfully established in the high fat feeding group,displaying impaired endothelial function especially in large arteries,and a corresponding endothelial function.G-CSF impacted the artery system.The impact was more significant in larger arteries.There were relationships in appearance and function between normal and arteriosclerosis endothelial cells,as well as in their reactions to G-CSF.Partâ…£Involvement of G-CSF in Chronic Artery Obstruction via the Possible Mechanism of Platelet Function ActivationObjective:To investigate the involvement of G-CSF in chronic artery obstruction via the possible mechanism of activation of platelet function during its application in cardiovascular diseases as a stem cell motivator.Methods:Sixteen male New Zealand white rabbits were randomly divided into the high fat feeding group and the control group.G-CSF was injected subcutaneously at a dose of 15ug/kg diluted to 0.5mL with normal sodium for a consecutive 5 days.PLT,MPV,PDW and P-LCR were assessed using blood cell counting instrument before and after drug.Platelet related index such as TXB2 and P-selectin were evaluated using ELISA.Platelet activation markers were validated using whole blood CD41-FITC/CD62P,CD63-PE fluorescent antibody double-labeled flow cytometry analysis.The alternation in the platelet aggregation function was assessed by platelet aggregation instrument.Results:The normal feeing group showed elevated index other than PLT after drug usage compared with basal conditions.The high fat feeding group showed change only in P-LCR.The high fat feeding group showed higher P-selectin and TXB₂ than the normal feeing group.The normal feeing group also showed a more significant change in P-selectin and TXB2 before and after drug usage[(16.39±2.23)ng/ml vs.(40.62±19.37)ng/ml(P＜0.05), (243.09±32.7)ng/ml vs.(473.87±80.17)ng/ml(P＜0.05)].Whole blood cell flow cytometer analysis displayed a large variation in platelet activation before and after G-CSF usage.The positive rate of CD62P was(0.2±0.01)%and(4.57±0.24)%, respectively(P＜0.05),and that of CD63 was(1.09±0.06)%and(7.14±0.62)%, respectively(P＜0.05).Platelet aggregation function showed significant difference in A_max before and after drug usage in the high fat feeding group[(33.51±0.55)%vs. (38.23±0.67)%(P＜0.05)],with no change in T_max and A₁(P＞0.05).All the three index showed significant difference in the normal feeding group before and after drug usage[(12.64±0.63)%vs.(36.9±0.73)%,(3.9±0.27)s vs.(2.19±0.19)s,(24.58±0.47)% vs.(45.58±0.96)%,P＜0.05].A_max and A₁ were significantly elevated in the high fat feeding group than the normal feeding group[(33.51±0.55)%vs.(12.64±0.63)%, (42.05±0.81)%vs.(24.58±0.47)%,P＜0.05].Conclusions:G-CSF caused the activation of platelet function on its highest peak,possibly due to stress reaction. Hyperglycemia has activated platelet before,so the motivation of G-CSF was blunted. P-LCR was an early predictor for platelet activation.Whole blood cell flow cytometer analysis was a quite sensitive tool for detecting platelet activation.Platelet aggregation rate assessment helped the evaluation of its activation.Based on the above studies,we deduce that activated platelet is possibly involved in the chronic obstructive artery diseases.Potential Applications and Innovations1.We have established a new animal model for chronic obstructive artery diseases with the advantages of convenience,high achievement ratio,easy to follow-up,and self-controlling,providing a sound experimental foundation for further researches.2.We realized the localization of artery constrictor made in China.The product is a domestic technology breakthrough and is under the application for national patent.3.Combined morphology observation with function detection,we made an overall description of the impact of G-CSF on arteries,especially on the local endothelium of chronic clogged vessels.4.Advanced methods such as Real-time PCR and scanning electron microscope were applied to make an objective description of the impact of G-CSF on vessels at the ultra structure and molecular level.5.The impacts of G-CSF on the artery system were further discussed,and the representative arteries of large,middle,and small size were picked up for discussion. This is especially meaningful for atherosclerosis,which is common in clinical practice.6.This is an early domestical study to report the impact of G-CSF on the function of platelet during the treatment of cardiovascular diseases as stem cell motivator,make an initial investigation on the possible involvement of thrombus into chronic artery obstruction.