The formation and death of macrophages and foam cells are one of the major factors that cause coronary heart disease (CHD). In our study, based on the Edinburgh Human Metabolic Network (EHMN) metabolic network, we built an enzyme network which was constructed by enzymes (nodes) and reactions (edges) called the Edinburgh Human Enzyme Network (EHEN). By integrating the subcellular location information for the reactions and refining the protein-reaction relationships based on the location information, we proposed a computational approach to select modules related to programmed cell death. The identified module was in the EHEN-mitochondria (EHEN-M) and was confirmed to be related to programmed cell death, CHD pathogenesis, and lipid metabolism in the literature. We expected this method could analyze CHD better and more comprehensively from the point of programmed cell death in subnetworks.
Coronary heart disease (CHD) is the narrowing or blockage of the coronary arteries, usually caused by atherosclerosis. Atherosclerosis is the buildup of plaques on the inner walls of the arteries, which can restrict blood flow to the heart muscle by physically clogging the artery or by causing abnormal artery tone and function [
Programmed cell death (PCD) is proposed to be death of a cell in pathological format, mediated by an intracellular program [
Traditional experimental research was through single gene or single compound to analyze CHD, and it could not provide the analysis at system level. Along with the rapid development of technology, the study about biological networks is increasingly providing valuable information on biological systems [
Therefore, in our paper, we presented a high-quality human metabolic enzyme network, Edinburgh Human Enzyme Network (EHEN), manually reconstructed based on Edinburgh Human Metabolic Network (EHMN) [
We used monocyte-derived macrophages from peripheral blood cultured in the absence or presence of oxidized LDL, baseline macrophages or foam cells. The macrophages were obtained from 15 subjects with subclinical atherosclerosis and a family history of CHD. Macrophages from 15 age and gender matched subjects with no atherosclerosis and no family history of CHD were used as control. We downloaded this expression data with the accession number of GSE9874 [
The Edinburgh Human Metabolic Network (EHMN) was reconstructed by integrating genome annotation information from different databases and metabolic reaction information from the literature [
Schematic diagram of enzyme network construction and protein location method. (a) Substrates and products of reactions are shown as blue circles, enzymes of reactions are shown as red hexagons, relationships of reaction are shown as purple mathematical symbols, edges of network are shown as black arrows, and the arrow pointing means the order of reactions. (b) The Gene ontology terms used for protein locations. The top locations are circled. Proteins are matched to GO terms and then backtracked to the selected locations through the hierarchical structure.
The enzyme protein location information in this work is extracted mainly from gene ontology (GO) [
We proposed a computational approach to select modules related to programmed cell death. All the disease and matched control samples were normalized simultaneously using the robust multiarray average (RMA) method [ Corrected Reporter enzymes of the EHEN were screened with The network functional modules based reporter enzymes were found by MCODE [ Gene ontology (GO) enrichment analysis was performed for each module from 6; select the modules related to programmed cell death. To repeat the above method in the 6 subnetworks, we got the reporter enzymes related network functional modules based on different subnetworks. Finally, we got reporter enzymes related network functional modules in the EHEN and six subnetworks.
In order to analyze the relationship between cell death and CHD, we select the functional modules related to programmed cell death for further research. GO functional enrichment analysis was applied for these modules using the Functional Annotation Tool in DAVID Bioinformatics Resources 6.7 (
Using the method described above in the network and subnetwork construction, all the human proteins coming from GO were classified into the chosen locations. By the described method, our constructed EHEN was separated into 6 subnetworks according to its localization information in our study, including cytoplasm (EHEN-C), mitochondria (EHEN-M), Golgi apparatus (EHEN-G), extracellular (EHEN-E), nucleus (EHEN-N), and endoplasmic reticulum (EHEN-R).
The input for network topological analysis to the calculation is the list of relationships of enzymes. Observing the degree distribution, characteristic path length, the connectivity, network diameter, the average and the maximum of the shortest path lengths, and clustering coefficient, the EHEN and subnetworks were found as scale-free following nearly a power law model (
From the reporter enzyme selection algorithms in the method part, we got reporter enzymes of overall and subnetwork, respectively. The numbers of reporter enzymes varied in different networks; the EHEN-C had the largest number of reporter enzymes, while the EHEN-R had only 2 selected reporter enzymes; the overall EHEN network stayed in the middle position (7 reporter enzymes), as listed in the Supplementary Material (Table S2: the reporter enzymes of overall and subnetwork). The degree and the clustering coefficient of the reporter enzymes had significant difference (
With the help of Cytoscape and its plug-ins, we selected out modules based reporter enzymes. From module division and annotation result of the EHEN and subnetworks by using Mcode, we gained functional modules of each network. Gene ontology (GO) enrichment analysis was performed for seven modules using DAVID, including 2 modules in the EHEN, 2 modules in the EHEN-C, and 3 modules in the EHEN-M (see Figure
Seven modules based on reporter enzymes.
CHD was caused by the plaques buildup of lipid, which was created by macrophages forming foam cells. We defined the classification principles based on CHD-related functions-CHD pathogenesis, lipid metabolism, and programmed cell death (Table
Module information of overall and part of subnetworks.
Network | Module count | RE count | Related function count | |||
---|---|---|---|---|---|---|
Basic metabolic | Lipid | CHD | Death | |||
EHEN | 2 | 4 | 32 | 18 | 9 | — |
EHEN-C | 2 | 6 | 40 | 22 | 22 | — |
EHEN-M | 3 | 5 | 51 | 13 | 35 | 15 |
Because coronary heart disease is caused by abnormal lipid metabolism, so the GO terms associated with lipid metabolism can be direct evidence between reporter enzymes and CHD. We were more interested in related programmed cell death reporter enzymes and modules.
The module 3 in mitochondria subnetwork (EHEN-M3) was a module which enriched to not only the basic metabolic function class, the related CHD pathogenesis, but also the related programmed cell death function class. It included 33 enzymes (2 reporter enzymes ec: 6.1.1.7 and ec: 6.1.1.17 and 31 others) (Figure
The module EHEN-M3 and the result of the functional enrichment. (a) The module 3 in EHEN-M. (b) The result of the functional enrichment.
Traditionally, there are several ways to research the mechanism of CHD. Our approach focused on CHD in the programmed cell death perspective on the systems biology level. We constructed a correlative network of enzymes considering the cascade. The differences from the macrophages to the foam cell between disease patients and normal controls were calculated according to the expression level. We proposed a computational approach to select 7 modules based on reporter enzymes (2 in the EHEN, 2 in the EHEN-C, and 3 in the EHEN-M). Most of the modules could be enriched to the basic metabolic function class, the related CHD pathogenesis, and the related lipid function class.
The module EHEN-M3 was related to programmed cell death in the EHEN-M. Functional and structural integrity of mitochondria was essential for the physiological function of the cardiovascular system. Accumulation of mitochondrial DNA mutations has been linked to ischaemic heart disease, cardiomyopathy, and atherosclerotic vascular disease. Mitochondria are known to regulate apoptotic and autophagic pathways that have been shown to play an important role in the development of cardiomyopathy and atherosclerosis [
It was reported that macrophages and foam cells were associated with CHD in the aspect of programmed cell death. Wang et al. reported that macrophage with lipid growing eventually formed foam cells until death. A large pool of bubble formation and programmed cell death eventually developed into a typical plaque [
Deficiency or alterations in metabolic functions were known to be involved in CHD. Enzyme proteins and chemical compounds were connected in metabolic networks. The reconstruction of the enzyme network could illustrate cascade relationships of enzymes. The selected reporter enzymes and modules from each subnetwork were more closely associated with CHD. We hope that the research would be more comprehensive with more data accumulation, such as posttranslational regulatory data. Considering from this aspect, enzyme marker and modules could be searched and be used in disease detection in the future.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Xu Jia, Wan Li, and Zhengqiang Miao contributed equally to this work.
This work was supported in part by the National Natural Science Foundation of China (Grant no. 61272388), the Natural Science Foundation of Heilongjiang Province (Grant no. F201237), the Science & Technology Research Project of the Heilongjiang Ministry of Education (Grant no. 12511271), and the Master Innovation Funds of Heilongjiang Province (Grant nos. YJSCX2012-209HLJ and YJSCX2012-224HLJ).