| BackgroundCalcific aortic valve disease (CAVD) is a chronic progressive disease, with aortic valve thickening and calcification, activity limitation, valvular stenosis and (or) insufficiency, causing angina, syncope and heart failure. With aging of society accelerating and human life expectancy increasing, the incidence of CAVD increased year by year, and has become the third major cardiovascular disease after coronary heart disease and hypertension. CAVD consists of two types, namely, aortic valve calcification (AVC) and calcific aortic valve stenosis (CAS). After investigation, heart valve disease in the United States over the age of 65 years or older population is 4.4～ 8.5%, and over the age of 75 years or older population is 11.7%～13.3%, while the calcific aortic valve stenosis over the age of 65 years or older population is 2.0～4.0%, and over the age of 75 years or older population is 2.8%. One-third of our elderly have echocardiographic or radiological evidence of aortic valve sclerosis (CAVS), an early and subclinical form of CAVD. However, even in middle age, approximately 10% exhibit CAVS by echocardiography. CAS is the most serious form of the development of CAVD.2% of individuals over age 60 suffer from CAS to the extent that surgery is required to preclude death once symptoms occur. Foreign reports indicate that among the aortic valve stenosis over the age of 65 years or older, there are 90% of the disease caused by aortic valve calcification. The latest research suggests that CAVD can result in higher cardiovascular accident death rate and total mortality, may be a new independent risk factor for coronary events. In addition, many other studies found that CAVD is closely related to the onset of carotid atherosclerosis. In 2012, aortic valve disease was directly responsible for> 14,000 deaths in the USA, making it the second-leading cause of cardiovascular mortality. Age, gender, smoking, high cholesterol, high blood pressure, type 2 diabetes increases the risk of the disease.Aortic valve calcification is mainly located in aortic side of valve, with two or three lobes involved at the same time. The lesions are mainly located in the basal part, along the aortic annulus deposition, gradually extended to the free edge of the valve, rarely involving the leaflet margins. Diseased valve became uneven thickening, with large number of calcium deposition. There are no cell components in the core of calcium deposition.With the change of population aging structure, proportion of CAVD is rising year by year. At present, there is no specific drug therapy for CAVD, and aortic valve replacement surgery is the only effective treatment options for patients with CAS. But surgical valve replacement also has its limitations, such as valve replacement is costly, and mechanical valve replacement surgery patients need long-term oral anticoagulation drugs, and the elderly often coexist diseases, increased the risk of bleeding in patients and some patients tend to have contraindications to anticoagulation; And the service life of the biological valve is limited. It can be a degenerative changes and can’t solve the problem for a lifetime. In addition, especially in patients with surgical extracorporeal circulation for elderly patients is high risk. So it is significant to develop an early intervention for CAVD, which is based on the study of the pathogenesis of the disease. However, the pathogenesis of CAVD is still not clear.Years CAVD is considered as a passive process of degeneration associated with aging and the result of the tissue fatigue or degradation. But recent research has shown that CAVD is not only a degeneration of the results, but also an active process involved multiple factors. It may be the reaction of valvular tissue to some of the initial factor, accompanied by chronic inflammation, inflammatory cells and lipid infiltration, cell differentiation, matrix remodeling, bone formation and calcification process. A number of studies indicate that both classical and non-classical NF kappa B pathway located in inflammatory center stage, activated by TNF alpha, play an important role in the inflammatory process of CAVD. Interleukin-6 (IL-6), transforming growth factor beta 1 (TGF-β1), matrix metalloproteinases (MMPs), heat shock protein 60 (Hsp60) and angiotensin II are overexpressed in calcific aortic valve, regulating valve’s inflammation, remodeling and angiogenesis.At present, the possible pathogenesis of CAVD is many, as:(1) Chronic inflammation:A number of studies have indicated that the calcification of aortic valve has the infiltration of chronic inflammatory cells, suggesting that CAVD is not merely a long-term leaflet wear, passive calcium deposition, and may is a pathogenic microorganism and mechanical flow shear stress of valvular skin damage, induce lipid deposition and by inflammatory cells and cytokines that mediate the formation of chronic activity of active inflammatory process. Cell apoptosis, osteopontin and bone matrix proteins involved in this process and form the ectopic calcification. (2) The genetic theory, studies have found that the occurrence of degenerative valvular disease has familial aggregation and genetic predisposition. Now confirmed that the vitamin D receptor gene, apolipoprotein E gene, eNOS G894T polymorphism is associated with the occurrence of degenerative valvular disease. (3) Calcium and phosphorus metabolism disorder:Histological findings revealed that degeneration of fibrous tissue and the deposition of calcium can be seen in the involvement valve. Further study found that vitamin D deficiency, lack of sex hormones and calcium loss lead to hyperparathyroidism, increasing secretion of parathyroid hormone, accelerating bone salt migration from bone to soft tissue, sedimentary in the valve and cause calcification. (4) Lipid abnormal accumulation:Patients with hyperlipidemia, especially in patients with high cholesterol, are more likely to have calcific valvular disease. Lipid abnormalities accumulation and a large number of foam cells may be found in the lesions of the valve, presumably lipid in valve aggregation, oxidation and play a role in valve fibroblasts, and finally cause valvular tissue degeneration, and lead to calcium salt deposition. (5) Extracellular matrix remodeling:Matrix metalloproteinase (MMP), mainly MMP2-3, MMP9 and MMP12 are higher expressed in calcific valves while this can not be detected in the normal valve, suggesting that the role of MMP in promoting matrix synthesis may play an important role in the calcific valvular disease. (6) Pressure loading mechanism:Calcific heart valve disease mainly involve the aortic valve and mitral valve. And aortic valve and mitral valve withstand the maximum pressure. Patients with very rare right heart valve calcification often have a right heart volume or pressure overload. Suggest that high velocity impact force and blood flow increased lead to flap ring injuries, causing tissue degeneration, hyperplasia of fibrous tissue, fatty infiltration or collagen fracture gap is formed, which is conducive to the deposition of calcium salt, accelerate the valvular process.(7) Aging degeneration:A number of studies have indicated that, with the increase of age, the incidence of calcific heart valve disease is greatly increased. At present, there is no definite boundary to the division of the age. Some researchers have found that 55 years of age or older are independent risk factors for the disease while other studies think of 60 years age or 50 years age. In view of the relationship between calcific valvular heart disease and aging, with the growth of age, in addition to the heart valve, other body organs and tissues also appear gradually calcium deposition and fibrous tissue degeneration. Therefore, we can speculate that the disease is the inevitable phenomenon of the human body in the aging process.Human aortic valve is composed of endothelial cells on the surface of the valve and the interstitial cells scattered in the interstitial layer. Valve interstitial cells are the main cellular components of the valve, which plays an important role in maintaining the normal physiological function of the valve. The study found valve interstitial cells can be induced by certain factors, such as oxidative stress, inflammatory cytokines, bone signaling, and transform into osteoblast like phenotype. Osteoblast like cells further secrete bone morphogenetic protein, alkaline phosphatase to promote ossification and calcification which play an important role in the occurrence and development of CAVD. Therefore, infiltration of inflammatory cells in the valve, secretion of inflammatory cytokines and valve interstitial cells transformation to muscle fibroblast and osteoblast like cells promote calcium nodules and bone formation, and this is an important pathophysiological mechanism of aortic valve calcification.Mast cells (MC) derive from hematopoietic stem cells, containing specific cytoplasmic granules and among them, storage of inflammatory mediators and cytokines such as chymotrypsin, trypsin, histamine, heparin and tumor necrosis factor alpha. Degranulation of mast cells can be induced by a variety of ways, in which the immunological mechanism of IgE dependent is a major way. MCs are widely distributed in the whole vascular tissue and play an important role in allergic reactions, innate immunity, acquired immunity and autoimmune diseases. MCs were rarely seen on the normal arterial wall, but significantly clustered in the atherosclerotic lesion. MCs are activated by a variety of mechanisms to release cytokines to induce the expression of vascular endothelial cell protease; release of chemokines to recruit inflammatory cells; release of proteases to promote the foam cells formation, angiogenesis, matrix degradation and vascular endothelial cell apoptosis and cytokines activation and thus involved in the occurrence and development process of atherosclerotic lesions. MC’s surface express Fc epsilon R I receptor and IgE can specificity combined with it to activate MCs resulting in the release of various inflammatory mediators and cytokines, leading to arterial vascular smooth muscle proliferation and tissue damage. CAVD and AS has a similar risk factors, in the pathogenesis of the disease also exist in common. However, there are a lot of patients with AS do not suffering from CAVD, suggesting that the pathogenesis of both is different.Immunoglobulin E (IgE) is the least content of the human antibody subtypes, mainly produced by plasma cells of mucous lamina located in nose pharynx, tonsil, bronchus, gastrointestinal and other. It’s human immune function (except in allergy and parasitic infection) for a long time has been unclear. IgE was cytophilic antibody, specifically bind with FcεR I on the mast cell membrane surface to make mast cells activated, and release a variety of inflammatory mediators and cytokines, resulting in artery vascular smooth muscle proliferation and tissue damage, promoting AS process. There have been studies indicated that the number of mast cells is closely related to the severity level of aortic calcification. Because there are common point between the pathogenesis of CAVD and AS, we speculate that IgE may also play an important role in the occurrence and development of CAVD.Based on the above theories and cognition, this study was to investigate the relationship between serum IgE levels and CAVD, and to investigate the possible molecular signaling mechanisms of IgE in human aortic valve interstitial cells in the induction of inflammation and ossification.Part I Alterated Serum Levels of Immunoglobulin E in Patients With Calcific Aortic Valve DiseaseObject Analysis the characteristic of serum immunoglobulin E (IgE) levels in patients with calcific aortic valve disease (CAVD) and to explore the alterated serum levels of immunoglobulin E (IgE) in patients with calcific aortic valve disease (CAVD).MethodCollect affiliated hospital of Southern Medical University 2012-2014 hospitalized patients’data who had serum immunoglobulin E (IgE) examination with age more than or equal to 46 and there are a total of 394 cases. The part of the crowd examined by ultrasound echocardiography diagram, among them 169 cases were clearly diagnosed aortic valve calcification in which 126 cases were male,43 female, namely calcific aortic valve disease group (CAVD group).225 cases were diagnosed non-aortic valve calcification by echocardiography, in which 159 cases were male,66 female, namely control group-non calcific aortic valve disease group (Non-CAVD group).Exclusion criteria:rheumatic heart disease with valvular heart disease, congenital heart malformation, valvular lesions caused by infection endocarditis, malignant tumor, allergic diseases (acute episode of asthma, allergic rhinitis, etc), autoimmune disease acute onset, arthritis acute attack (clear history of arthritis and/or rheumatoid factor positive), with other tissue organs obvious infection diseases.Register the object of study of relevant clinical data:age, inpatient number, age, gender, contact phone, ID number, immunoglobulin E (IgE), C reactive protein (CRP), Procalcitonin (PCT), triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), etc.All cases in the group get completely thoracic echocardiography examination, including M type, two-dimensional and Doppler echocardiography. According to the acoustic cardiogram test results into calcific aortic valve disease group and non-calcific aortic valve disease group. Judgement standard of calcific aortic valve disease are as follows:Except for aortic valve disease caused by rheumatic heart disease, infective endocarditis, congenital heart disease and other diseases, Aortic valve:Aortic valve limitations or diffuse echo enhancement [where disc or ring of echo intensity (EV) is more than or equal to the same section of the aortic root wall echo, that EV is more than or equal to Eaor, think valve leaf or flap ring calcification and valvular thickening (more than or equal to 2.0 mm), unrestricted movement of the leaflets, cross valve flow rate< 2.5m/s, valve area≥3cm2 is defined as aortic valve calcification; transvalvular blood flow rate≥ 2.5 m/s, valve area＜3cm2 is defined as aortic stenosis.All datas were analyzed by taking statistical software SPSS 20.0, parameters in line with the normal distribution described with mean+/-standard deviation, parameters in not in line with the normal distribution described with median. Test of homogeneity of variance use Levene method. T test was used to compare with two independent samples according with normal distribution. The comparison between non normal distribution data use Mann-Whitney U test of non parametric test. Two independent samples were compared by chi square test. Multi factor Logistic regression model was established according to the significance of t test and non parametric test, and the relationship between each index and CAVD was analyzed. All tests were two-sided test, P＜0.05 think the difference was statistically significant.Result(1) Mann-Whitney U test of Non parametric test showed that there was no significant difference of age between the CAVD group and the Non-CAVD group (Z value was-1.810, P>0.05). The chi square test indicated that there was no significant difference of gender between the two groups (X20.730, P>0.05). The Mann Whitney U test showed that the serum level of IgE in the CAVD group were significantly higher than those in the Non-CAVD groups (113.30 IU/ml vs 63.76 IU/ml, P< 0.05); There were also significant differences in the level of C reactive protein between the two groups (14.80 vs mg/L 6.60 mg/L,<0.05 P); In addition, there was no significant difference in the level of triglyceride and procalcitonin between the two groups. Two independent samples t test showed that there was no significant difference between the two groups in total cholesterol and low density lipoprotein cholesterol (P>0.05).(2) Taking CAVD as the dependent variable, the meaningful research indicators above as argument to further analyse. The results showed that the level of IgE、CRP in CAVD group were still significantly higher than that in Non-CAVD group (P<0.05).ConclusionThrough compare the serum levels of IgE between CAVD group and Non-CAVD group, we find that serum levels of IgE in the CAVD group were significantly higher than those in Non-CAVD group. In the multivariate logistic regression equation, IgE levels in CAVD group were still higher than that of Non-CAVD group, suggested that serum levels of IgE is independent biochemical indexes for CAVD and IgE may play an important role in the pathogenesis of CAVD.Part II A preliminary study on the Mechanism of Immunoglobulin E in calcific aortic valve diseaseObjectTo preliminarily explore the mechanism of immunoglobulin E (IgE) in calcific aortic valve disease (CAVD).Method1. A total of 24 patients’aortic valve who have taken aortic valve replacement operation were collected from the Department of thoracic surgery of affiliated hospital of Southern Medical University during 2015-2016. According to echocardiography and clinical data,10 cases were diagnosed as calcific aortic valve disease namely the experimental group, and 14 cases were non-calcific aortic valve disease, namely control group.2. Aortic valve interstitial cells (AVIC) isolated from human aortic valve tissue.①Registed the relevant clinical data of each study:name, in-hospital number, age, sex, telephone number, ID number, echocardiography results, etc. The samples were divided into 2 groups according to the clinical data and the results of echocardiography, calcific aortic valve disease group and non-calcific aortic valve disease group. The valve specimens were paraffin sections, and take toluidine blue staining to see the presence of mast cells.②With 50ug/ml IgE and 50ug/ml IgE+NF-kappa B inhibitor treated human aortic valve interstitial cells for 24 hours. After 24 hours, collected cell lysates, immunoblotting was used to detect the expression level of ICAM-1, BMP-2 and ALP.ResultToluidine blue staining, immunofluorescence and immunohistochemistry showed that there are mast cells and IgE in CAVD group while no mast cells and IgE in Non-CAVD group. After IgE stimulating human aortic valve interstitial cells, the expression levels of BMP-2, ICAM-1 and ALP detected by Western blotting were increased when compared with those without IgE stimulation. With NF- kappa B inhibitor, the expression level of ICAM-1, BMP-2 and ALP were reduced compared with IgE alone stimulation.ConclusionThe mast cells and IgE contents in CAVD group were higher than those in Non-CAVD group. IgE can stimulate human aortic valve interstitial cells expressing and secreting inflammatory mediators and ossification mediators. IgE stimulates human aortic valve interstitial cells’inflammation and ossification reaction by NF-kappa B signaling pathway. |
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