Transforming Growth Factor Binding Protein 2 As A Marker Of Heart Failure In The Preliminary Study

Despite considerable advances in the treatment of Cardiovascular disease, it remains the leading cause of mortality and morbility in the world. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. In previous studies, we found latent TGF-(3binding protein2(LTBP-2) that were seldom studied were upregulated in end-stage failing human myocardium through genomics screening. LTBP-2is an extracelluar matrix (ECM) protein, belongs to the fibrillin-LTBP gene family. Human LTBP-2is expressed mostly in the lung and to a lesser extent in the liver, skeletal muscle placenta and heart. Unlike the other LTBPs, relatively little is known about the functional role of LTBP-2, partly due to that further elucidation of the functional role of LTBP-2is limited by the fact that deletion of LTBP-2in mice leads to embryonic lethality. It was reported that LTBP-2can function in cell adhesion, artery injury repair and assembling of elastic fiber.To confirm the previous results Of genomic screening, we evaluated the cardiac level of LTBP-2in end-stage heart failure patients with histological analyses and ELISA, and we also studied changes of expression of LTBP-2during ventricular remodeling following myocardial infarction with mice models. We observed the effect of LTBP-2expression in cardiomyocyte by varied cytokines. The results showed that TGF-β1can significant promote the LTBP-2expression in the pilot study. TGF-β1, as an important cytokines, involved various pathophysiological processes of cardiovascular disease, including fibrosis and ventricular remodeling. LTBP-2may involve in ventricular remodeling through the regulation of TGF-β1. We explored TGF-β1induced LTBP-2in neonatal rat cardiomyocytes with its underlying signal transduction pathway. We also tested the hypothesis that human serum LTBP-2levels are increased in patients with heart failure with reduced ejection fraction (HFREF) and analyzed potential value of serum LTBP-2as a biomarker to confirm and rule out the presence of HFREF in patients with dyspnea.Main contents of this study including:Part I The expression of LTBP-2in the cardiac tissues of patients with end-stage heart failure (HF) and myocardial infarction (MI) model, and effect of TGF-β1on LTBP-2in neonatal rat ventricular cardiomyocytes (NRVMs) and its underlying signal transduction pathway.1. LTBP-2expression was elevated in the cardiac tissues of patients with end-stage HF. For histological analyses and ELISA of cardiac LTBP-2, human left ventricular (LV) myocardial samples were obtained from34patients diagnosed with end-stage HF due to dilated cardiomyopathy (DCM, n=19), ischemic cardiomyopathy(ICM, n=8), and arrythmogenic right ventricular cardiomyopathy (ARVC, n=7) during cardiac surgery at the time of heart transplantation and8non-failing control subjects died from accident with no history of heart disease. In the light microscope examination by hematoxylin-eosin (HE) staining, compared to normal hearts, varying degree of interstitial fibrosis in failing hearts due to ICM and DCM, and fibrofatty or fatty replacement in failing hearts due to ARVC were observed (Figure1A). In the immunohistochemical analyses using specific antibody against LTBP-2, strongly immunoreactive LTBP-2was observed in the failing hearts due to ICM, DCM and ARVC in contrast to normal control hearts with few and weak immunoreactive LTBP-2. The diffuse staining pattern of LTBP-2protein in failing hearts might reflect the fact that they are secreted. ELISA further confirmed that the median level of cardiac LTBP-2were significantly increased in heart failure patients (n=34) compared with normal control subjects (n=8)(974pg/mg v.s.193pg/mg, p<0.001).2. LTBP-2expression was elevated in the cardiac tissues of mice with MI. Myocardial infarction was created in SD rats by ligation of the left anterior descending coronary artery. The rats were randomly divided into2groups:sham group and MI group. Animals were sacrificed at1day,3day,1week,2weeks,4weeks,8weeks after operation and the left ventricular tissues were isolated for determination of LTBP-2by ELISA. Compared with the sham group, LTBP-2protein was regulated after MI, and especially significant in1day and1week. HE staining showed the model of MI was successfully established.3. Primary cultured cardiomyocytes were obtained from neonatal rats by enzymatic digestion method. LTBP-2gene and its protein expression were examined by Real-time PCR, Western blot analysis and immunocytochemistry. In order to further study the signal transduction pathway of TGF-β1, inhibitors of TGF-β1signaling pathways were used to inhibit the effect of TGF-β1and to observe the expression change of LTBP2. The stimulation effect of TGF-β1was dose-dependent, LTBP-2gene expression began to increase after2ng/ml (p<0.05) of stimulation, peaked at5ng/ml (p<0.05) and reached a plateau after10ng/ml (p<0.05). The stimulation effect of TGF-β1was also time-dependent, with the stimulation time extension, the expression of LTBP-2was increased, peaked at12h and declined at24h (p<0.05). Immunocytochemistry showed that TGF-β1increased LTBP-2levels. Further signaling pathway study demonstrated that TGF-β1induced expression of LTBP-2in cardiomyocytes via ERK pathway and PI3K pathway.Part Ⅱ Evaluation of the diagnostic capability of LTBP-2in patients with HFREF.For determination of serum LTBP-2and NT-proBNP, blood samples were acquired from a cohort of133enrolled consecutive patients with dyspnea referred to out-patient clinic or admitted into our institution (Fuwai Hospital, Beijing) between September2007and December2008. Another cohort of age-and gender-matched subjects (n=87) were enrolled as healthy controls without clinical risk factors and echocardiographic abnormalities. Left ventricular ejection fraction (LVEF) was used to divide the patients into two groups according to the severity of the ventricular impairment based on assessment by echocardiography. Patients were diagnosed as HFREF if the LVEF was<40%(n=67), or heart failure with preserved ejection fraction (HFPEF) if the LVEF was>40%(n=66). Determination of the levels of LTBP-2and NT-proBNP in three groups:Normal group, HFPEF group and HFREF group. Linear regression analysis was performed to examine the correlation between two variables. Receiver operating characteristic (ROC) curves were plotted to assess the diagnostic accuracy of LTBP-2and NT-proBNP and their combination.ELISA showed that both LTBP-2and NT-proBNP elevated by comparing Normal group, HFPEF group and HFREF group. In the three groups, the level of LTBP-2was365.8pg/ml,587.1pg/ml and857.6pg/ml and the level of NT-proBNP was109.1pg/ml,126.5pg/ml and229.3pg/ml. Linear correlation analyses showed no correlation between serum LTBP-2and NT-proBNP levels (R=0.04, p=0.26). Both serum LTBP-2and NT-proBNP levels were negatively correlated with left ventricular ejection fractioa (R=-0.31, p=0.001and R=-0.32, p<0.001, respectively). ROC analyses showed similar diagnostic performance for LTBP-2and NT-proBNP (AUC=0.67and0.68, respectively) in separating HFREF from HFPEF. The AUC of combined LTBP-2and NT-proBNP showed a little better performance but was not significantly different from the AUC of LTBP-2(0.73v.s.0.67, p=0.11), and NT-pro BNP (0.73v.s.0.68, p=0.31) alone.In summary, we demonstrated a significant elevation of cardiac LTBP-2in heart failure patients with LVEF<40%. ERK1/2and Akt were shown to be signaling molecules responsible for elevated LTBP-2. Serum LTBP2levels may be used to confirm and rule out the presence of HFREF in patients with dyspnea. Our findings suggest that LTBP-2might involve in cardiac remodeling and act as a promising biomarker in heart failure. More exploration of LTBP-2is needed to confirm the findings. Heart failure is a complex clinical syndrome with high morbidity and mortality. Coronary heart disease and hypertension are major underlying causes of heart failure. Other frequent underlying conditions include valvular heart disease and idiopathic dilated cardiomyopathy. It is difficult to predict who will develop heart failure in response to myocardial injury. Impairment of cardiac function activates compensatory neurohormonal mechanisms, which at a later stage may accelerate progression of heart failure. The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in the processes of heart failure. In response to sustained activation of the RAAS, the angiotensin-II receptors are deregulated in the human failing heart. Several downstream intracellular signaling effectors are overexpressed and activated in tandem with cardiac hypertrophy. Racial differences in occurrence and outcome of heart failure suggest a genetic contribution to the pathophysiology of left ventricular remodeling and heart failure. In addition to the discovery of disease-causing (rare) mutations, common variants in genes that encode neurohormonal, adrenergic, intracellular and interstitial proteins have been demonstrated to modulate the course and consequences of heart failure. The angiotensin-converting enzyme (ACE), a key enzyme in the renin-angiotensin-aldosterone system (RAAS) which plays an important role in the regulation for cardiac function, has got great attention about its genetic role in HF.So far, several case-control studies have investigated whether the ACE I/D polymorphism is associated with the risk of HF. The conclusions of these studies were inconsistent because of small studies and heterogeneous samples. A small-scale meta-analysis in a large systematic review concluded that there was no significant association between ACE I/D polymorphism and ischemic heart failure, however it is limited by including only ischemic etiology of HF and a small sample size with only five case-control studies. We conducted a meta-analysis of studies relating the ACE I/D polymorphic variant to the risk of HF.Main contents of this study including:We systematically searched PUBMED, EMBASE, previous reviews and reference lists from identified articles with no language limit published up to June2011related HF and genetic polymorphisms. We used the following search terms:(angiotens in-convert ing enzyme or ACE) and (polymorphism or mutation) and (cardiovascular disease or heart failure).The meta-analysis included case-control genetic association studies fulfilling accordingly inclusion criteria. All literature searches were independently reviewed by two professional co-workers to identify studies met the inclusion criteria. Differences were resolved by consensus. We used a standard reporting form to extract data from each study that we included. We collected information on first author, year of publication, country, journal, racial descent of study population, demographics, number of cases and controls for each genotype, genotyping methods, etiology of HF (ischemic HF, IHF or dilated HF, DHF), and confirmation of diagnosis. Where allele frequencies were not given, they were calculated from the corresponding genotype frequencies of the case and the control groups. We conducted a meta-analysis to investigate the association between ACE I/D and HF for the allele contrast (D vs. I), the recessive (DD vs. ID and II), dominant (DD and ID vs. Ⅱ), and additive (DD vs. DI) models. Subgroup analyses were performed based on racial/ethnic descent (whites and East Asian) and the etiology of HF (IHF and DHF). A total of1978articles were identified by the combined search of databases (PUBMED, EMBASE and Cochrane Library) and manual approach (searching previous studies cited in previous reviews, and reference lists from identified articles) of case-control studies, of which17case-control studies satisfied the inclusion criteria and were eventually included in the meta-analysis. In the17case-control studies, a total of5576participants were included in the meta-analysis, including2453cases with HF and3123controls. There was evidence of heterogeneity between studies in the main-analyses (DD vs. ID/II, P=0.02; DD/ID vs. II, P=0.000and D vs. I, P=0.01) and the risk for DHF (DD/ID vs. Ⅱ, P=0.02and D vs. I, P=0.04). The main analysis for investigating the association between the ACE I/D polymorphic variant and the risk of HF showed no association under the four genetic models. In sub group analysis by etiology, we also did not find a significant association between the ACE I/D polymorphic variant and the risk of IHF and DHF. In subgroup analysis by ethnicity, no significant associations between ACE variant and HF were found in both Whites and East Asian.In summary, this meta-analysis showed no evidence of the value of ACE I/D polymorphism as a marker for the increased risk of HF. Large-scale prospective and case-control studies are still required. Future studies should focus on gene-gene and gene-environment interactions to shed light on the genetics of HF.