| BackgroundAtherosclerosis(AS)is a common vascular disease.In the Western developed countries,50%of the population’s death is directly or indirectly related to AS,which has become the leading cause of cardiovascular disease worldwide.AS mainly involves the intima of large and medium-sized vessels and the pathological basis is lipid metabolism disorder.The main characteristics of AS are accumulation of large amounts of lipids,plaque formation and arterial lumen stenosis.AS is a disease caused by multiple factors,with complicated pathogenesis and unclear specific mechanismVascular calcification is a common pathological manifestation of atherosclerosis,hypertension,diabetes,vascular injury,chronic kidney disease and aging.Studies have shown that 90%of patients with coronary atherosclerotic heart disease have varying degrees of calcification in their arteries,and its severity may be directly associated with vascular stenosis in diseases characterized by AS.According to the different calcification location,the vascular calcification can be divided into the vascular intimal calcification,the vascular middle membrane calcification and the vascular outer membrane calcification.Vascular intimal calcification is associated with AS plaque formation and is a risk factor for early plaque formation and an increased risk of plaque rupture.Vascular middle membrane calcification is an important pathological manifestation of vascular injury in patients with end-stage renal disease and type 2 diabetes mellitus.Arterial calcification is an active,cell-regulated process occurring during osteogenesis and includes the transition of vascular smooth muscle cells(VSMCs)to osteoblasts and to the crystallization and precipitation of hydroxyapatite salt.Runt-related transcription factor 2(Runx2),bone morphogenetic protein 2(BMP2)and alkaline phosphatase(ALP)interact and influence each other,leading to a process similar to that of osteoblast-like differentiation.However,the mechanisms of atherosclerotic vascular calcification formation and regulation have not been fully elucidated.Carbonic anhydrase 1(CA1)is a member of the carbonic anhydrase(CA)family that reversibly catalyzes the hydration of CO2 to form HCO3-,which then rapidly binds to calcium ions to form calcium carbonate.We have found that CA1 expression is specifically upregulated in the synovial membrane in patients with ankylosing spondylitis.The most distinctive pathological manifestations of ankylosing spondylitis are inflammation of the hip and spinal joints,hyperosteogeny,joint fusion and fibrosis.We then found that CA1 could promote joint calcification,ossification and joint fusion by accelerating calcium carbonate deposition.Additionally,our group found that CA1 was highly expressed in breast carcinoma tissues and in blood from patients with breast cancer,leading to the calcification of the tumor tissue,inhibition of apoptosis and migration of tumor cells.Thus,the above results indicate that CA1 not only stimulates the biological calcification process,but also promotes the occurrence and development of pathological calcification.Other research groups have gradually found that CAs may play an important role in AS.As shown in the study by Oksala et al.,CA2 and CA12,members of the CA family,were highly expressed in human atherosclerotic plaques and might be associated with osteoclast-like cells of a mononuclear cell lineage in patients with advanced AS and were involved in plaque remodeling.Ando et al.examined abdominal aortic aneurysm using proteomics and detected an abundant CA1 autoantigen,suggesting an important role for CAI in the formation of this lesion.Based on their findings and the results reported by our group,we hypothesize that CA1 plays an important role in the AS process by stimulating tissue calcificationAcetazolamide(AZ)and methazolamide(MTZ)are CA inhibitors.MTZ and AZ are sulfonamide derivatives and are clinical drugs used in the treatment of glaucoma.MTZ is an improved version of AZ with a 60%stronger inhibitory effect on CA than AZ and a relatively low adverse reaction rate.As shown in our previous studies,the induction of calcification in human osteosarcoma Saos-2 cells and murine mammary adenocarcinoma 4T1 cells upregulated CA1 expression.The above studies suggested that AZ could effectively inhibit calcification of saos-2 cells and 4T1 cells.Pierce and Hall et al.found that CA activity can increase bone resorption and CA inhibitor MTZ can inhibit bone resorption through culture experiments of newborn mice in vitro.Nolan et al.found that MTZ,AZ,ethoxzolamide,diclofenamide and other CA inhibitors could inhibit joint deterioration in the rat model of adjuvant arthritis and alleviate joint edema in the rats.They believed that CA inhibitors could prevent and treat chronic inflammation by inhibiting bone absorption.Treatment of these cells with AZ not only reduced CA1 expression but also suppressed cell calcification.Our clinical study showed that MTZ was an effective treatment for patients with active ankylosing spondylitis by suppressing CA1 expression and joint fusion.The above studies indicate that CA inhibitors MTZ and AZ can achieve therapeutic effect by inhibiting the expression of CA1 and the calcification process it regulates.Therefore,we believe that CA inhibitors may play a therapeutic role on AS and its vascular calcification process by inhibiting the expression of CA1.Based on our previous research and the findings of others,the purpose of this study was to explore the role and mechanism of CA1 on AS vascular calcification and the therapeutic effect of CA inhibitor MTZ on AS.This study was divided into two parts.In the first part,the expression of CA1 in AS tissues of human aorta was firstly detected,and the relationship between CA1 expression level and AS lesions were observed.Then,the ApoE-/-mouse AS model was established to detect the expression of CA1 in the aortic tissues of model mice.The relationship between CA1 expressionand calcium salt deposition was observed to analyze the relationship between CA1 and AS calcification.Meanwhile,in this study,the CA inhibitor MTZ was applied in the mouse AS model to observe the effect of MTZ on the formation of AS plaques and its therapeutic effect on AS in vivo.Vascular smooth muscle cells are the core sites of vascular calcification,and the transformation of vascular smooth muscle cells to osteoblastic phenotype is the core link of vascular calcification.However,the regulatory mechanism of CA1 in vascular calcification is still unclear.Therefore,in the second part of this study,vascular smooth muscle cells were selected as experimental subjects to study the role of CA1 in vascular smooth muscle cell calcification.In the second part of this study,rat vascular smooth muscle cells(rat VSMCs)were cultured to induce calcification in vitro,and CA1 expression was observed after calcification.Meanwhile,anti-CA1 siRNA and CA inhibitor AZ were applied to treat VSMCs to inhibit the expression of CA1,and the role of CA1 in VSMC calcification was analyzed and determined.The proliferation,migration and apoptosis of VSMCs were observed by interfering with the expression of CAI.The second part of the study aims to explore the mechanism of CA1 on AS vascular calcification through cell experiments in vitro.MTZ is a clinical drug,but no sterile MTZ for cell culture is available.Sterile AZ for cell culture experiments is commercially available,but clinical AZ is not.We thus treated the mouse model with MTZ in the first part and cultured the VSMCs with AZ in the second part of this studyPart 1:High expression of CA1 in AS tissues and effect of MTZ on mouse AS modelMethods1.In order to study the expression of CA1 in human aortic tissues,14 samples of human aortic tissues were collected through cardiac surgery in the department of cardiac surgery at Shandong Provincial Qianfoshan Hospital.These included healthy aortic specimens from the control group(heart transplant donors)(n=7)and human aortic AS tissues(Patients with aortic aneurysm and aortic dissection were selected as the study subjects.Patients with both AS and vascular calcification symptoms were screened through case inquiry and imaging detection data.Aortic tissue specimens were obtained after cardiac surgery as the specimens of AS experimental group)(n=7).Total protein were extracted and the expression of CA1 protein in human aortic tissue samples was detected by Western blot.In this study,human aortic tissue chip(Alenabio,xi ’an,China)was used to detect the expression of CA1 protein in healthy human aortic tissues(n=8)and AS tissues(n=8)by immunohistochemistry.Von Kossa staining was used to detect calcium salt deposition in healthy human aortic tissues(n=8)and AS tissues(n=8),and the relationship between calcium salt deposition and CA1 protein expression was analyzed2.In order to study whether CA1 plays an important role in AS calcification and whether the CA inhibitor methazolamide(MTZ)has a therapeutic effect on AS,we successfully established an AS model by administration of a high-fat diet to apolipoprotein E(ApoE-/-)mice.AS models were set up,with feeding MTZ in week 12(MTZ treatment group),or at the beginning of the feeding high fat forage(MTZ preventive treatment group).Total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-c),high-density lipoprotein cholesterol(HDL-c)and nitric oxide(NO)levels in the mouse serum were measured using kits.Inflammatory cytokines were measured using flow cytometry.Western blot and immunohistochemistry were used to detect the expression of CA1 in mouse aortic tissues.Hematoxylin-eosin staining was used to observe the changes of aortic structure and morphology.Mouse AS plaques were observed using Sudan IV staining and Oil Red O staining.Calcium deposition in the mouse aorta was observed using Von Kossa staining.Results1.CA1 protein was highly expressed in human AS tissues detected by Western blot and immunohistochemistry.Von Kossa staining showed that there was a large amount of calcium salt deposition in AS tissue of human aorta,and CA1 was highly expressed in the calcium salt deposition site.These results indicate that CA1 may be closely related to the pathogenesis of AS calcification.2.The administration of a high-fat diet to mice for 21 weeks led to the successful establishment of the AS model.After 21 weeks of high-fat diet,serum lipid test data showed that compared to the control group,serum TC,TG and HDL-c levels of mice were significantly increased,while HDL-c and NO levels were significantly decreased in the AS model group.Compared to the AS model group,the serum TC,TG and LDL-c levels were significantly decreased,while HDL-c and NO levels were significantly increased in MTZ treatment group.Serum levels of TC,TG and LDL-c were decreased,HDL-c and NO were significantly increased in the mice of MTZ preventive treatment group compared to the mice of the AS model group.The difference in the levels of TC,TG,and HDL-c between the MTZ treatment group and the MTZ preventive treatment group was not statistically signifcant,but the difference in the LDL-c levels was statistically signifcant.Compared to the AS model group,serum concentrations of IL-6,IFN-y,GM-CSF,TNF-α,CXCL1 and MCP-1 were significantly decreased in the mice that received MTZ treatment or MTZ preventive treatment.The above results showed that MTZ had a significant therapeutic effect on AS mice,and MTZ could reduce the production of some serum inflammatory cytokines3.After 21 weeks of high-fat diet feeding,Western blot and immunohistochemistry results showed that CA1 was highly expressed in AS tissues of mice aorta,and MTZ could inhibit the expression of CA1.In the aortas of the AS group,clear concentric AS plaques,accompanied by massive foam cells that accumulated inside the plaques,were observed.Large amounts of cholesterol crystals were present.The smooth muscle layer was relaxed and structurally disrupted.Inflammatory cells had accumulated.The intima was thickened and protruded into the lumen,leading to stenosis of the lumen.Vascular intima had a large amount of calcium salt deposition.These results indicated that the AS model had been successfully built.Compared to the AS model group,the MTZ treatment group and the MTZ preventive group had significantly less AS plaques,less foam cells and cholesterol accumulation,more regular smooth muscle layer,decreased inflammatory cell aggregation,and enlarged lumens,and the MTZ preventive group had the weakest AS degree.The results of Sudan IV staining and Oil Red O staining showed the MTZ treated mice had reduced AS plaque areas and fat accumulation.Von Kossa staining showed that there were large deposits of dark brown calcium salt in the intima of mice in the AS model group.Compared to the AS model group,the MTZ treatment group and the MTZ preventive treatment group could hardly see the obvious calcium salt deposition,and the CA1 high expression region of immunohistochemistry mainly appeared in the calcium salt deposition site.These results demonstrated that CA1 expression and CA1-mediated calcification are significantly associated with AS progression.MTZ treatment can significantly reduce the degree of AS in mice.Conclusions1.Significantly increased expression of CA1 protein was detected in human and mouse aortic AS tissues,and there was high expression of CA1 in the calcium salt deposition site,indicating that CA1 is strongly associated with AS vascular calcification2.In the mouse AS model,the CA inhibitor MTZ reduced serum levels of TC,TG and HDL-c,increased serum levels of HDL-c and NO,reduced the production of inflammatory factors IL-6,IFN-γ,GM-CSF,TNF-α,CXCL1 and MCP-1 in the mouse AS model,and reduced the formation of AS plaques,proving that MTZ has the effect of treating AS3.In the mouse AS model,high expression of CA1 was related to AS vascular calcification,and MTZ inhibited AS vascular calcification.CA1 is the target of MTZ,indicating that MTZ may inhibit AS vascular calcification by inhibiting the expression of CA1 and CA1-mediated calcification,thus playing a role in the treatment of AS.Part 2:Role and mechanism of CA1 in vascular smooth muscle cell calcification Methods1.In order to detect the relationship between CA1 and vascular smooth muscle cell(VSMC)calcification,rat vascular smooth muscle cells were cultured with or without Acetazolamide(AZ),a CA inhibitor with similar chemical structure to MTZ,and was induced to calcification with β-glycerophosphate(β-GP).The calcification was measured using Alizarin Red-S(AR-S)staining and cetylpyridinium chloride assay.Real-time PCR was used to detect the expression levels of runt-related transcription factor 2(Runx2),alkaline phosphatase(ALP)and bone morphogenetic protein 2(BMP2)in order to study cell ossification.The expression of CA1 before and after calcification was detected by Western blot.The expression level of inflammatory cytokines in cell culture medium was detected by flow cytometry.2.In order to detect the effects of CA1 expression on the proliferation,migration and apoptosis of VSMCs,VSMCs were transfected with anti-CA1 small interfering ribonucleic acid(siRNA)and Allstars siRNA using the HiPerFect Transfection Reagent(QIAGEN,Germany).Western blot analysis was used to detect the CA1 expression level.Cell counting kit-8 assay was used to observe the effect of CA1 on cell proliferation,Transwell assay was used to determine the effect of CA1 on cell migration and flow cytometry was used to investigate the effect of CA1 on cell apoptosis.3.In order to study the effect of CA1 expression on calcification and the production of inflammatory cytokines,VSMCs were also transfected with anti-CA1 siRNA and β-GP was added to induce calcification.Western blot was used to detect the expression of CA1 protein in VSMCs after transfected with anti-CA1 siRNA.AR-S staining was used to observe the formation of calcification nodules.mRNA expression of Runx2,ALP and BMP2 was detected by Real-time PCR.The expression of inflammatory cytokines in cell culture medium was detected by flow cytometry.Results1.VSMC calcification was significantly increased upon induction with β-GP,AR-S staining showed a large number of calcification nodules.Western blot and Real-time PCR showed that the expression of CA1 and other ossification-related genes Runx2,ALP and BMP2 were significantly increased.After AZ treatment,the calcification nodules were significantly reduced,the expression of CA1 was decreased,and mRNA expression of ossification marker protein was also significantly decreased.The results of flow cytometry showed that the expression level of inflammatory cytokines in cell culture medium was significantly reduced after AZ treatment.The above results indicated that CA1 was closely related to VSMC calcification,and inhibit the expression of CA1 could inhibit the calcification,ossification and inflammatory cytokines(IL-6,IFN-γ,GM-CSF,and TNF-α)production of VSMCs.This suggests that CA1 may stimulate the expression of the inflammatory factors IL-6,IFN-γ,GM-CSF,and TNF-α of VSMCs2.The results of Cell Counting kit-8,Transwell experiment and flow cytometry showed that anti-CA1 siRNA significantly suppressed cell proliferation and migration,and promoted apoptosis.These results suggest that CA1 may stimulate the proliferation and migration and inhibit apoptosis of VSMCs3.VSMC calcification was induced by β-GP,and inhibition of CA1 expression could significantly reduce the production of calcification nodules.Western blot and Real-time PCR results showed that inhibition of the expression of CA1 decreased the expression of ossification-related genes Runx2,ALP and BMP2.This indicates that CA1 expression has a regulatory effect on VSMC calcification.The results of flow cytometry showed that the expression level of inflammatory cytokines in cell culture medium decreased significantly after anti-CA1 siRNA transfection.The above results further indicated that anti-CA1 siRNA could suppress the production of VSMC calcification,ossification and inflammatory cytokines(IL-6,IFN-γ,GM-CSF,and TNF-α)production.This suggests that CA1 may stimulate VSMC calcification by increasing the expression of inflammatory factors.Conclusions1.β-GP induced VSMC calcification,and CA1 expression was significantly increased after calcification.The application of CA inhibitors AZ and anti-CA1 siRNA could inhibit the expression of CA1 and inhibit cell calcification,indicating that the expression of CA1 may stimulate VSMC calcification2.In the VSMC calcification model,the application of CA inhibitors AZ and anti-CA1 siRNA could inhibit the production of inflammatory factors IL-6,IFN-y,GM-CSF,and TNF-α in cell culture medium,indicating that CA1 may stimulate VSMC calcification by regulating the expression of inflammatory factors IL-6,IFN-y,GM-CSF,and TNF-α.3.The results of Cell Counting kit-8 experiment,Transwell experiment and apoptosis experiment proved that CA1 may stimulate proliferation and migration and inhibite apoptosis of VSMCs.To sum up,CA1 is highly expressed in the AS tissues of the human aorta and in the aortic AS tissues of the animal model,and CA1 expression and CA1-mediated calcification are significantly associated with AS progression.Methazolamide plays a therapeutic role on AS by inhibiting the expression of CA1 and CA1-mediated calcification.Inhibition of CA1-mediated calcification may be the key point in the treatment of atherosclerosis.Methazolamide represents a potential treatment for atherosclerosis. |
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