Inflammatory markers and coronary heart disease

PURPOSE OF REVIEW: Despite changes in lifestyle and the use of effective pharmacologic interventions to lower cholesterol levels, coronary heart disease remains the major cause of morbidity and mortality in the developed world. Cholesterol screening fails to identify almost 50% of those individuals who will present with acute coronary syndromes. Recent evidence from laboratory and prospective clinical studies demonstrates that atherosclerosis is not simply a disease of lipid deposition, but rather is an inflammatory process with highly specific cellular and molecular responses. The clinical utility of inflammatory markers has been examined in a variety of atherothrombotic diseases. Because C-reactive protein is highly stable in stored frozen samples, and automated and robust analytical systems for its measurement are available, it has become the most widely examined inflammatory marker. RECENT FINDINGS: C-reactive protein has consistently been shown to be a useful prognostic indicator in acute coronary syndromes and is a strong predictor of future coronary events in apparently healthy individuals. In addition, C-reactive protein can identify individuals with normal lipid levels who are at increased risk for future coronary events. Because drugs such as aspirin and statins reduce inflammatory risk, C-reactive protein has the potential to guide the use of these therapies in high-risk individuals for primary prevention. SUMMARY: C-reactive protein may have a role in global risk assessment for primary prevention and in targeting those patients who will benefit from anti-inflammatory therapies. In addition, it may also be a good prognostic indicator in patients with acute coronary syndromes.     

Effects of transdermal estrogen on levels of lipids, lipase activity, and inflammatory markers in men with prostate cancer

Androgen deprivation therapy (ADT) for prostate cancer is now used in earlier disease stages and as adjuvant treatment. Recognizing and reducing the toxicity of this therapy, including worsened lipid levels and cardiovascular disease (CVD) risks, has become an important clinical concern. Oral estrogen therapy induces hypogonadism and mitigates many side effects of ADT, but has a high thrombosis risk. Transdermal estrogen therapy (TDE) has a lower thrombosis risk than oral estrogen and may improve CVD risk compared with ADT. This prospective pilot study of 18 men with androgen-independent prostate cancer receiving ADT measured effects of TDE on lipid and inflammatory CVD risk factors before and after 8 weeks of TDE (estradiol 0.6 mg/day). During treatment, estradiol levels rose 17-fold; total cholesterol, LDL cholesterol, and apolipoprotein B levels decreased. HDL2 cholesterol increased, with no changes in triglyceride or VLDL cholesterol levels. Dense LDL cholesterol decreased and LDL buoyancy increased in association with a decrease in HL activity. Highly sensitive C-reactive protein levels and other inflammatory markers did not worsen. Compared with ADT, short-term TDE therapy of prostate cancer improves lipid levels without deterioration of CVD-associated inflammatory markers and may, on longer-term follow-up, improve CVD and mortality rates.     

NMR analysis of cardiac troponin C-troponin I complexes: effects of phosphorylation.

Phosphorylation of the cardiac specific amino-terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N-terminal cardiac troponin I proteins, cardiac troponin I(33-80), cardiac troponin I(1-80), cardiac troponin I(1-80)DD and cardiac troponin I(1-80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1-80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N-terminal domain of cardiac troponin I primarily makes contact with the C-terminal domain of cardiac troponin C. The nonphosphorylated cardiac specific amino-terminus, cardiac troponin I(1-80), was found to make additional interactions with the N-terminal domain of cardiac troponin C.     

High affinity peptides for the recognition of the heart disease biomarker troponin I identified using phage display

Troponin I is a specific and sensitive clinical biomarker for myocardial injury. In this study we have used polyvalent phage display to isolate unique linear peptide motifs which recognize both the human and rat homologs of troponin I. The peptide specific for human troponin I has a sequence of FYSHSFHENWPS and the peptide specific for the rat troponin I has a sequence of FHSSWPVNGSTI. Enzyme‐linked immunosorbent assays (ELISAs) were used to evaluate the binding interactions, and the two phage‐displayed peptides exhibited some cross‐reactivity, but they were both more specific for the troponin I homolog they were selected against. The binding affinities of the phage‐displayed peptides were decreased by the presence of complex tissue culture media (MEM), and the addition of 10% calf serum further interfered with the binding of the target proteins. Kinetic indirect phage ELISAs revealed that both troponin I binding peptides were found to have nanomolar affinities for the troponin proteins while attached to the phage particles. To our knowledge, this is the first example of isolation and characterization of troponin I binders using phage display technology. These new peptides may have potential utility in the development of new clinical assays for cardiac injury as well as in monitoring of cardiac cells grown in culture. Biotechnol. Bioeng. 2010. 105: 678–686. © 2009 Wiley Periodicals, Inc.     

心肌肌钙蛋白Ⅰ的分子印迹电化学生物传感器

应用分子印迹技术,以邻苯二胺和对苯二酚为功能单体,心肌肌钙蛋白Ⅰ(cTnI)为模板分子,在pH 7.0磷酸盐缓冲液中,利用循环伏安法在玻碳电极表面聚合形成了分子印迹膜.该分子膜对cTnI有特异性识别作用,在0.01～2.00 μg/mL的范围内,cTnI的浓度与氧化峰电流的变化呈线性关系,检测下限为2 ng/mL,响应时间为15 min.该分子印迹传感器具有制备简单、特异性及稳定性好等优点.     

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.     

心肌型脂肪酸结合蛋白胶体金检测试纸条的制备和初步应用

目的应用胶体金免疫层析法制备检测全血或血清样本中心肌型脂肪酸结合蛋白（H-FABP）的检测试纸条,用于急性心肌梗塞（AMI）的早期辅助诊断。方法采用柠檬酸三钠还原法制备胶体金,标记鼠抗心肌型脂肪酸结合蛋白单克隆抗体,喷于玻璃纤维膜上制成胶体金结合物垫,将另一株鼠抗心肌型脂肪酸结合蛋白单克隆抗体和抗鼠二抗分别包被检测线和质控线,组装成试纸条进行灵敏性、特异性、精密性、稳定性及临床样品检测。结果该试纸条的检测灵敏度为10ng／mL,15min内可判定结果;与肌钙蛋白I、C反应蛋白、肌酸激酶、人心肌肌红蛋白无交叉反应。检测240份临床标本,与临床诊断结果进行配对分析,阳性符合率95．83％、阴性符合率100％、总符合率97．92％。结论制备的H-FABP检测试纸条有良好的灵敏性、特异性,可用于早期AMI的辅助诊断。     

The interaction between beef cardiac troponin T and troponin I as demonstrated by ultraviolet absorption difference spectroscopy, circular dichroism, and gel filtration.

The specific interaction of bovine cardiac troponin T with troponin I has been demonstrated at a 1:1 molar ratio by absorption difference spectroscopy, near and far ultraviolet circular dichroism, and gel filtration chromatography. The maintenance of the sulfhydryl groups of both proteins in the reduced state was essential in order to demonstrate interaction between cardiac troponin I and troponin T using the aforementioned methodology. Carboxamido-methylated troponin I and troponin T samples were prepared by reaction with iodoacetamide. Spectrophotometric titration of the two proteins with 2-chloromercurinitrophenol and amino acid analysis of their carboxamidomethylated derivatives revealed that cardiac troponin I possesses two cysteine residues while cardiac troponin T has one. The modified troponin T possesses properties identical to those of the native molecule. The modification of troponin I is accompanied by an increase in secondary structure and a loss in ability to interact with troponin T at 0.5 M NaCl ionic strength. However, at 0.3 M NaCl the modified troponin I was shown by gel filtration chromoatography to interact very weakly with troponin T. On the other hand, the modified troponin I interacts with troponin C in a manner identical to the native protein, indicating that the troponin T interaction domain of the molecule is distinct from that region which interacts with troponin C.     

Protein kinase C phosphorylation of cardiac troponin I or troponin T inhibits Ca2(+)-stimulated actomyosin MgATPase activity.

Effects of troponin phosphorylation on Ca2(+)-stimulated MgATPase activity of bovine cardiac actomyosin were examined. Phosphorylation by protein kinase C of troponin I and troponin T subunits in troponin or troponin-tropomyosin complex resulted in a decreased Ca2(+)-stimulated MgATPase activity in reconstituted actomyosin, and this effect was reversed by subsequent dephosphorylation by protein phosphatase 1. It was further observed that protein kinase C phosphorylation of either troponin I or troponin T subunits led to a similar inhibition of Ca2(+)-stimulated actomyosin MgATPase activity. In all cases, EC50 values (concentrations causing 50% stimulation) for Ca2+ were not appreciably affected by troponin phosphorylation by protein kinase C. Data from phosphorylation site analysis suggests that phosphorylation of threonine 144 in troponin I and possibly threonine 280 or threonine 199 in troponin T might be important for the observed decrease of Ca2(+)-stimulated actomyosin MgATPase. It is suggested that inhibition of actomyosin MgATPase caused by protein kinase C phosphorylation of troponin I and/or troponin T represents a new mechanism that can account for in part the reported negative inotropic effect of phorbol esters on various cardiac preparations.     

The phosphorylation of troponin I from cardiac muscle.

1. Troponin I isolated from fresh cardiac muscle by affinity chromatography contains about 1.9 mol of covalently bound phosphate/mol. Similar preparations of white-skeletal-muscle troponin I contain about 0.5 mol of phosphate/mol. 2. A 3':5'-cyclic AMP-dependent protein kinase and a protein phosphatase are associated with troponin isolated from cardiac muscle. 3. Bovine cardiac 3':5'-cyclic AMP-dependent protein kinase catalyses the phosphorylation of cardiac troponin I 30 times faster than white-skeletal-muscle troponin I. 4. Troponin I is the only component of cardiac troponin phosphorylated at a significant rate by the endogenous or a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. 5. Phosphorylase kinase catalyses the phosphorylation of cardiac troponin I at similar or slightly faster rates than white-skeletal-muscle troponin I. 6. Troponin C inhibits the phosphorylation of cardiac and skeletal troponin I catalysed by phosphorylase kinase and the phosphorylation of white skeletal troponin I catalysed by 3':5'-cyclic AMP-dependent protein kinase; the phosphorylation of cardiac troponin I catalysed by the latter enzyme is not inhibited.     

Interleukin-18 as a Therapeutic Target in Acute Myocardial Infarction and Heart Failure

Interleukin 18 (IL-18) is a proinflammatory cytokine in the IL-1 family that has been implicated in a number of disease states. In animal models of acute myocardial infarction (AMI), pressure overload, and LPS-induced dysfunction, IL-18 regulates cardiomyocyte hypertrophy and induces cardiac contractile dysfunction and extracellular matrix remodeling. In patients, high IL-18 levels correlate with increased risk of developing cardiovascular disease (CVD) and with a worse prognosis in patients with established CVD. Two strategies have been used to counter the effects of IL-18:IL-18 binding protein (IL-18BP), a naturally occurring protein, and a neutralizing IL-18 antibody. Recombinant human IL-18BP (r-hIL-18BP) has been investigated in animal studies and in phase I/II clinical trials for psoriasis and rheumatoid arthritis. A phase II clinical trial using a humanized monoclonal IL-18 antibody for type 2 diabetes is ongoing. Here we review the literature regarding the role of IL-18 in AMI and heart failure and the evidence and challenges of using IL-18BP and blocking IL-18 antibodies as a therapeutic strategy in patients with heart disease.     

香丹注射液对急性冠脉综合征患者外周血C-反应蛋白、氨基末端脑钠肽及心肌酶水平的影响

目的:探讨香丹注射液治疗急性冠脉综合征血清C-反应蛋白、氨基末端脑钠肽以及心肌酶学的影响。方法:收集我院心内科收治的急性冠脉综合征患者66例,随机分为试验组和对照组,各33例。对照组予以阿司匹林片和硫酸氢氯吡格雷片治疗,试验组在对照组基础上予以香丹注射液治疗。观察并比较两组患者的临床疗效、血清肌钙蛋白T、C-反应蛋白、氨基末端脑钠肽、心肌酶学、不良反应以及心脏事件发生情况。结果:试验组临床总有效率高于对照组(P0.05)。治疗后两组肌钙蛋白T、C反应蛋白以及氨基末端脑钠肽水平降低,且试验组低于对照组(P0.05)。治疗后两组肌酸激酶同工酶、肌酸激酶、天门冬氨酸转移酶以及乳酸脱氢酶水平降低,且试验组低于对照组(P0.05)。试验组心绞痛发作次数、心肌缺血时间以及室性早搏次数较对照组相比显著降低(P0.05)。两组不良反应比较,差异无统计学意义(P0.05)。术后1个月内试验组心脏事件发生率显著低于对照组,差异有统计学意义(P0.05)。结论:香丹注射液治疗急性冠脉综合征的疗效显著,抑制炎症反应,安全性较高,降低心脏事件发生率,适宜临床应用推广。     

Sex-specific changes in platelet aggregation and secretion with sexual maturity in pigs.

Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2-3 mo) and sexually mature (adult; 5-6 mo) male and female pigs (n=8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 microM) and collagen (6 microg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.     

Detection of a novel sense-antisense RNA-hybrid structure by RACE experiments on endogenous troponin I antisense RNA

Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this "hybrid RNA." Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and antisense troponin I RNA in vivo, which we discuss in the article.     

The dynamic range problem in the analysis of the plasma proteome

One of the greatest challenges in analyzing the plasma proteome is the wide range of concentration of different proteins. The current study examines the range of protein concentration for 18 proteins measured over a year in a clinical laboratory to provide data on pathological extremes in protein concentrations. The complete measured range, from upper limits for albumin to lowest values for thyroid-stimulating hormone (TSH), represented more than 10 logs of molar abundance. A number of plasma proteins measured in the clinical laboratory varied over a concentration range spanning more than 4 logs, and limits of detection of clinical assays were inadequate to assess full concentration ranges of several proteins. Considering reported values from studies using higher sensitivity assays suggest that plasma concentrations of prostate-specific antigen (PSA), human chorionic gonadotropin (hCG), and cardiac troponin I vary by more than 7 logs. All of the plasma proteins measured in the present study represent secretory proteins or highly expressed components of specific tissues. Thus, the dynamic range for these components is likely to greatly underestimate the total range of protein concentration in the plasma proteome.     

Hypothyroidism and Its Rapid Correction Alter Cardiac Remodeling

The cardiovascular effects of mild and overt thyroid disease include a vast array of pathological changes. As well, thyroid replacement therapy has been suggested for preserving cardiac function. However, the influence of thyroid hormones on cardiac remodeling has not been thoroughly investigated at the molecular and cellular levels. The purpose of this paper is to study the effect of hypothyroidism and thyroid replacement therapy on cardiac alterations. Thirty Wistar rats were divided into 2 groups: a control (n = 10) group and a group treated with 6-propyl-2-thiouracil (PTU) (n = 20) to induce hypothyroidism. Ten of the 20 rats in the PTU group were then treated with L-thyroxine to quickly re-establish euthyroidism. The serum levels of inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6) and pro-fibrotic transforming growth factor beta 1 (TGF-β1), were significantly increased in hypothyroid rats; elevations in cardiac stress markers, brain natriuretic peptide (BNP) and cardiac troponin T (cTnT) were also noted. The expressions of cardiac remodeling genes were induced in hypothyroid rats in parallel with the development of fibrosis, and a decline in cardiac function with chamber dilation was measured by echocardiography. Rapidly reversing the hypothyroidism and restoring the euthyroid state improved cardiac function with a decrease in the levels of cardiac remodeling markers. However, this change further increased the levels of inflammatory and fibrotic markers in the plasma and heart and led to myocardial cellular infiltration. In conclusion, we showed that hypothyroidism is related to cardiac function decline, fibrosis and inflammation; most importantly, the rapid correction of hypothyroidism led to cardiac injuries. Our results might offer new insights for the management of hypothyroidism-induced heart disease.     

Effects of troponin I phosphorylation on conformational exchange in the regulatory domain of cardiac troponin C.

Conformational exchange has been demonstrated within the regulatory domain of calcium-saturated cardiac troponin C when bound to the NH2-terminal domain of cardiac troponin I-(1-80), and cardiac troponin I-(1-80)DD, having serine residues 23 and 24 mutated to aspartate to mimic the phosphorylated form of the protein. Binding of cardiac troponin I-(1-80) decreases conformational exchange for residues 29, 32, and 34. Comparison of average transverse cross correlation rates show that both the NH2- and COOH-terminal domains of cardiac troponin C tumble with similar correlation times when bound to cardiac troponin I-(1-80). In contrast, the NH2- and COOH-terminal domains in free cardiac troponin C and cardiac troponin C bound cardiac troponin I-(1-80)DD tumble independently. These results suggest that the nonphosphorylated cardiac specific NH2 terminus of cardiac troponin I interacts with the NH2-terminal domain of cardiac troponin C.     

The effects of two types of Western diet on the induction of metabolic syndrome and cardiac remodeling in obese rats

Metabolic syndrome (MetS) include obesity as a critical feature and is strongly associated with risk of cardiovascular disease (CVD). Insights into mechanisms involved in the pathophysiology of these clinical manifestations are essential for the development of therapeutic strategies. Thus, Western diets (WD) have been widely employed in diet-induced obesity (DIO) model. However, there are variations in fat and sugar proportions of such diets, making comparisons challenging. We aimed to assess the impact of two types of the WD on metabolic status and cardiac remodeling, to achieve a DIO model that better mimics the human pathogenesis of MetS-induced CVD. Male Wistar rats were distributed into three groups: control diet, Western diet fat (WDF), and Western diet sugar (WDS) for 41 weeks. Metabolic and inflammatory parameters and cardiac changes were characterized. WDF and WDS feeding promoted higher serum triglycerides, glucose intolerance, and insulin resistance, while just WDF presented inflammation in adipose tissue. WDF-fed rats showed increased catalase activity and malondialdehyde (MDA) and carbonyl protein levels, suggesting cardiac oxidative stress, while WDS-fed rats only raised MDA. Both WD equally elevated protein expressions involved in lipid metabolism, but only WDF downregulated the glycolysis pathway. Furthermore, the mechanical myocardial function was impaired in obese rats, being more relevant in WDF. In conclusion, both WD effectively triggered MetS features, although inflammation was detected just on the WDF-fed animals. Moreover, the WDF promoted a more pronounced functional, metabolic, and oxidative cardiac disorder, suggesting to be an adequate model for studying CVD in the scenario of MetS.     

Enhanced death signaling in ozone-exposed ischemic-reperfused hearts

Although numerous advancements made in the field of human health have resulted in reduced deaths due to cardiovascular diseases (CVD), many patients with cardiac disease show no established risk. Therefore, other unknown factors may be responsible for the pathophysiology of CVD. Out of 350,000 sudden cardiac deaths each year in the United States, 60,000 deaths have been related to air pollution, suggesting a detrimental role of environmental pollutants in the development of CVD. The present study tested our hypothesis that chronic ozone exposure enhances the sensitivity to ischemia–reperfusion (I/R) injury in isolated perfused hearts. Sprague-Dawley rats were continuously exposed for 8 h/day for 28 and 56 days to filtered air or 0.8 ppm ozone. Isolated hearts were subjected to 30 min of global ischemia followed by 60 min of reperfusion. Cardiac function after I/R measured as left ventricular developed pressure (LVDP), +dP/dt, –dP/dt, and left ventricular end diastolic pressure (LVEDP) was significantly decreased and increased respectively in ozone-exposed I/R hearts compared to I/R hearts exposed to filtered air. The enhanced sensitivity to I/R injury upon ozone exposure was associated with increased myocardial TNF-α levels and lipid peroxidation and decreased myocardial activities of superoxidase dismutase (SOD) and IL-10. These data suggest that ozone-induced sensitivity to myocardial I/R injury may be due to promoting levels of oxidative stress as well as inflammatory mediators.