| Vascular calcification is a complex pathological change in which hydroxyapatite minerals and amorphous calcium deposit are actively deposited in the vascular system mediated by a variety of cells.Compared with others,the vascular calcification in diabetic patients is usually more frequent and severe because of the hidden onset and different predilection sites,which significantly increases the occurrence of adverse prognosis such as cardiovascular events and amputations.Until now,the treatment method of the advanced calcification is limited: most of the marketed therapeutic drugs such as pyrophosphate have been found to have serious side effects;and surgical treatment such as interventional spin grinding and laser ablation may cause coronary artery perforation and embolization of downstream small blood vessels.In view of the clinical significance and the bottleneck of late treatment,there is a great need to establish a more appropriate and earlier diagnosis method for vascular calcification in order to take timely clinical intervention.At present,imaging detection methods such as flat-scan CT,X-ray photography,ultrasound and IVUS are more common for clinical diagnosis of vascular calcification.Flat-scan CT is mainly used for the evaluation of the coronary artery calcification with high accuracy,but not the peripheral large vessel;IVUS is the gold standard for calcification detection,but it is not suitable for the population examination because of the invasiveness;ultrasound is only applicable to the superficial blood vessel calcification due to its powerless to deep blood vessels;the calcification index method using X-ray is only used in the evaluation of abdominal aortic calcification due to the lack of natural segmentation marks similar to vertebrae in other parts.Due to the limitations of the above methods,the screening rate of the vascular calcification is still low,and the diagnostic criteria have not covered all the sites of vascular calcification such as the lower extremity arteries below the knee,nor can take a systemic vascular calcification detection,which is the difficult clinical status of vascular calcification detection.Therefore,we expect to develop a new calcification diagnosis methods or biological targets to provide a new entry point for earlier prevention,diagnosis,and treatment of diabetic vascular calcification.In this study,we discussed the diagnostic methods of vascular calcification in diabetic patients from the two directions of imaging and serum immunology.In terms of imaging,we proposed a spiral CT-based lower limb vascular calcification evaluation index(LCI)and confirmed its accuracy and effectiveness in clinical and pathological experiments;in terms of serum immunology,we confirmed the effectiveness of a serum component that might be used for the diagnosis of the systemic vascular calcification of diabetes,namely carboxymethyllysine(CML),by clinical trials,combining with an in-depth study on its specific mechanism of action.The contents of each part are summarized as follows:Part I: A new calcification index based on multi-slice spiral CT to evaluate the calcification of lower extremity arteries in DF patientsObjective:Due to inadequacy of the existing diagnosis methods of the diabetic lower limb vascular calcification,this study is expected to estimate a new method named Lower limb arterial Calcification Index(LCI),and verify its effectiveness in the evaluation of the lower extremity arterial calcification.Methods:A total of 254 diabetic foot patients who were hospitalized in the affiliated hospital of Jiangsu University from January 2016 to January 2019 were selected and divided into 6 groups according to the Wagner diagnostic criteria.We tried to establish a new calcification index of the lower extremity arteries in followings:dividing the lower extremity blood vessels below the knee(anterior tibial artery /posterior tibial artery / fibular artery)into six parts referring to the Bollinger's vascular segmentation method;Scoring each vascular segment referring to the Kaupplila's Abdominal Aortic Calcification Index(ACI)scoring criteria;Summing the score of each vascular segment to get a total LCI score.Further,the correlation between the LCI and the DF grade,also the correlation between the LCI and the risk of amputation during the follow-up experiment was studied;the consistency of the LCI and pathological vascular calcium content was investigated using the amputated blood vessels of patients with DF grade 5.Aiming at the problem of insufficient consideration of calcification within the plaque in the calcification index method,the regulation effect of the vascular stenosis(VS)on the calcium content and LCI was studied as well.Results:We have successfully established an evaluation system for the calcification index of the lower extremity arteries,which can effectively and quantitatively evaluate the calcification of the lower extremity arteries of DF patients,with a certain effect of amputation risk prediction.The results of clinical trials showed that the LCI score increased significantly with the DF grade;in the 2-year follow-up experiment with the end of the amputation event,the average LCI score of the amputated patient was much higher than that of others;in the pathological experiment,there was a significant consistency between the LCI and calcium content in the lower limb arteries provided by amputated patients(W = 0.373,P <0.05),and the VS is an effective regulator of the calcium content and LCI with a critical point of 0.907.Conclusions:LCI is an effective and easy method for evaluating the calcification of lower limb arteries with a certain effectiveness of amputation risk prediction.Part II: Associations between serum concentration of N?-carboxymethy lysine and carotid artery calcification in Diabetes Mellitus patients: A clinical researchObjective:At present,imaging detection methods are more common for clinical diagnosis of vascular calcification,with a lack of systemic examination method for early diagnosis.This study aimed the early immunological diagnosis method for vascular calcification,and investigated the effectiveness of a potential marker-glycotoxic metabolite serum N?-carboxymethyl lysine(CML)which has been proved as a strong promoting factor on calcification in the previous study.Methods:A total of 506 patients in the Affiliated Hospital of Jiangsu University from2016.1-2017.6 were included,of these,148 patients have type 1 DM,and 191 patients have type 2 DM.All patients underwent color Doppler ultrasonography to detect bilateral carotid arteries.The severity of carotid calcification was evaluated according to 0 to 8 points,with 1 to 4 points being mild,and 5 to 8 points being severe.Multivariate logistic regression analysis was conducted to determine the independent predictors of different degrees of carotid calcification in type 1 and type 2 DM patients.Results:In the high calcification group and the low calcification group,the levels of CML,IMT and low density lipoprotein in diabetic patients were higher than those in the control group(P<0.05),and the type 2 DM group was higher than the type 1 DM group(P<0.05).Multivariate logistic regression analysis demonstrated that CML were independent predictors for carotid calcification in type 2 DM patients(CML,OR2.025,95% CI 1.368-2.996;P < 0.05),while in type 2 DM patients,CML were also independent predictors for carotid calcification(CML,OR 2.485,95% CI 1.481-4.171;P < 0.05).Follow-up suggested that a significant increase in CML levels during the follow-up period may be related to adverse events.Conclusions:CML may be an independent predictor of carotid artery calcification at different degrees in patients with type 1 and type 2 diabetes,and to some extent predict the occurrence of diabetic cardiovascular and cerebrovascular events.Part III: Study of NFATc1 on its effect in CML leaded calcification of mice vascular smooth muscle cellsObjective:Activating T cell nuclear factor 1(NFATc1)can promote the differentiation and function of osteoblast.We investigate whether NFATc1 is involved in the osteoblastic phenotype and functional differentiation of the vascular smooth muscle cells,which is the main participant of CML leaded ectopic calcification.Methods:Primary vascular smooth muscle cells were extracted and identified from the aorta of C57 BL / 6J mice using tissue affixing method.First,the primary smooth muscle cells of the 3-5th passages were divided into the control group,and the cells added with N?-carboxymethyllysine(CML)(10 umol/ L)were divided into the stimulation group.Western Blot was used to detect the NFATc1 expression in the two groups.Then,the stimulation group was further divided into two groups,one of which was not treated as an interference-free group,and the other group was interferenced using small interfering RNA(si RNA)to knockout NFATc1 stably as an NFATc1 silence group.Then,q RT-PCR and Western Blot was used to verify the interference efficiency.Finally,Von kossa staining and alizarin red staining were used to detect the calcification of the control group,non-interference group,and NFATc1 silence group for comparison.Results:Compared with the control group,Western Blot result showed a significant increase in the expression of NFATc1 in the stimulation group;calcification test result showed that the calcification in the non-interference group was significantly higher than that in the control group,and was decreased in the NFATc1 silence group substantially.Conclusions:NFATc1 participates in the progress of CML leaded smooth muscle vascular calcification in mice,that can provide an important theoretical basis for CML as a serum calcification marker.It is also expected to be a new target to interfere with the progress of the vascular calcification promoted by CML to help the treatment of the vascular calcification,but the detailed mechanism needs to be further studied as well. |
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