Identification of Risk Genes Associated with Myocardial Infarction-Big Data Analysis and Literature Review.

Acute myocardial infarction occurs when blood supply to a particular coronary artery is cut off, causing ischemia or hypoxia and subsequent heart muscle destruction in the vascularized area. With a mortality rate of 17% per year, myocardial infarction (MI) is still one of the top causes of death globally. Numerous studies have been done to identify the genetic risk factors for myocardial infarction, as a positive family history of heart disease is one of the most potent cardiovascular risk factors. The goal of this review is to compile all the information currently accessible in the literature on the genes associated with AMI. We performed a big data analysis of genes associated with acute myocardial infarction, using the following keywords: "myocardial infarction", "genes", "involvement", "association", and "risk". The analysis was done using PubMed, Scopus, and Web of Science. Data from the title, abstract, and keywords were exported as text files and imported into an Excel spreadsheet. Its analysis was carried out using the VOSviewer v. 1.6.18 software. Our analysis found 28 genes which are mostly likely associated with an increased risk for AMI, including: PAI-1, CX37, IL18, and others. Also, a correlation was made between the results obtained in the big data analysis and the results of the review. The most important genes increasing the risk for AMI are lymphotoxin-a gene (LTA), LGALS2, LDLR, and APOA5. A deeper understanding of the underlying functional genomic circuits may present new opportunities for research in the future.

In vivo multidimensional CRISPR screens identify Lgals2 as an immunotherapy target in triple-negative breast cancer.

Immune checkpoint inhibitors exhibit limited response rates in patients with triple-negative breast cancer (TNBC), suggesting that additional immune escape mechanisms may exist. Here, we performed two-step customized in vivo CRISPR screens targeting disease-related immune genes using different mouse models with multidimensional immune-deficiency characteristics. In vivo screens characterized gene functions in the different tumor microenvironments and recovered canonical immunotherapy targets such as Ido1. In addition, functional screening and transcriptomic analysis identified Lgals2 as a candidate regulator in TNBC involving immune escape. Mechanistic studies demonstrated that tumor cell-intrinsic Lgals2 induced the increased number of tumor-associated macrophages, as well as the M2-like polarization and proliferation of macrophages through the CSF1/CSF1R axis, which resulted in the immunosuppressive nature of the TNBC microenvironment. Blockade of LGALS2 using an inhibitory antibody successfully arrested tumor growth and reversed the immune suppression. Collectively, our results provide a theoretical basis for LGALS2 as a potential immunotherapy target in TNBC.

Galectin-2 in Health and Diseases.

Galectin-2 is a prototype member of the galactoside-binding galectin family. It is predominately expressed in the gastrointestinal tract but is also detected in several other tissues such as the placenta and in the cardiovascular system. Galectin-2 expression and secretion by epithelial cells has been reported to contribute to the strength of the mucus layer, protect the integrity of epithelia. A number of studies have also suggested the involvement of galectin-2 in tissue inflammation, immune response and cell apoptosis. Alteration of galectin-2 expression occurs in inflammatory bowel disease, coronary artery diseases, rheumatoid arthritis, cancer, and pregnancy disorders and has been shown to be involved in disease pathogenesis. This review discusses our current understanding of the role and actions of galectin-2 in regulation of these pathophysiological conditions.

Syndecan 4, galectin 2, and death receptor 3 (DR3) as novel proteins in pathophysiology of preeclampsia.

INTRODUCTION: Preeclampsia has the highest rate of obstetric morbidity and mortality. METHODS: We recruited 21 women with preeclampsia and 27 women with uncomplicated pregnancies. We used a quantitative protein macroarray that allowed for analysis of 40 proteins. RESULTS: We found a statistically significant increase in the concentration of DR3, LIF and a significant decrease of VEGF, PlGF, syndecan-4 and galectin-2, in the plasma of women with preeclampsia. CONCLUSIONS: There are no previous studies assessing syndecan 4, galectin 2, and DR3 concentrations in women with preeclampsia; Our results indicate these proteins are new factors that play important roles in the immunological pathomechanism of preeclampsia.

Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth.

Colorectal cancer is the third leading cause of cancer-related deaths in the United States and the third most common cancer in men and women. Around 20% colon cancer cases are closely linked with colitis. Both environmental and genetic factors are thought to contribute to colon inflammation and tumor development. However, the genetic factors regulating colitis and colon tumorigenesis remain elusive. Since reactive oxygen species (ROS) is vitally involved in tissue inflammation and tumorigenesis, here we employed a genome-wide CRISPR knockout screening approach to systemically identify the genetic factors involved in the regulation of oxidative stress. Next generation sequencing (NGS) showed that over 600 gRNAs including the ones targeting LGALS2 were highly enriched in cells survived after sublethal H2O2 challenge. LGALS2 encodes the glycan-binding protein Galectin 2 (Gal2), which is predominantly expressed in the gastrointestinal tract and downregulated in human colon tumors. To examine the role of Gal2 in colitis, we employed the dextran sodium sulfate (DSS)-induced acute colitis model in mice with (WT) or without Lgals2 (Gal2-KO) and showed that Gal2 deficiency ameliorated DSS-induced colitis. We further demonstrated that Gal2-KO mice developed significantly larger tumors than WT mice using Azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal cancer model. We found that STAT3 phosphorylation was significantly increased in Gal2-deficient tumors as compared to those in WT mice. Gal2 overexpression decreased the proliferation of human colon tumor epithelial cells and blunted H2O2-induced STAT3 phosphorylation. Overall, our results demonstrate that Gal2 plays a suppressive role in colon tumor growth and highlights the therapeutic potential of Gal2 in colon cancer.

Expression, S-Nitrosylation, and Measurement of S-Nitrosylation Ratio of Recombinant Galectin-2.

S-nitrosylation, which involves the coupling of an NO group to the reactive thiol of Cys residue(s) in a polypeptide, is an important posttranslational modification detected in a variety of proteins. Here, we present the S-nitrosylation of recombinant galectin-2 (Gal-2) using S-nitrosocysteine and the measurement of the molecular ratio of S-nitrosylation of Cys residues in the Gal-2 protein.

Characterisation and functional comparison of single-CRD and multidomain containing galectins CgGal-2 and CgGal-3 from oyster Crassostrea gigas.

Galectins are ß-galactoside binding lectins that play crucial roles in innate immunity in vertebrates and invertebrates through their conserved carbohydrate-recognition domains (CRDs). In the present study, single- and four-CRD-containing galectins were identified in oyster Crassostrea gigas (designated CgGal-2 and CgGal-3). The open reading frames (ORFs) of CgGal-2 and CgGal-3 encode polypeptides of 200 and 555 amino acids, respectively. All CRDs of CgGal-3 include two consensus motifs essential for ligand-binding, and a novel motif is present in CgGal-2. Pathogen-associated molecular pattern (PAMP) profiles were determined for recombinant rCgGal-2 and rCgGal-3, and rCgGal-2 displayed low binding affinity for PAMPs, while rCgGal-3 bound various PAMPs including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN) with relatively high affinity. Furthermore, rCgGal-2 and rCgGal-3 exhibited different microbe binding profiles; rCgGal-2 bound to Gram-negative bacteria (Escherichia coli and Vibrio vulnificus) and fungi (Saccharomyces cerevisiae and Pichia pastoris), while rCgGal-3 bound to these microbes but also to Gram-positive bacteria (Micrococcus luteus). In addition, rCgGal-3 possessed microbial agglutinating activity and coagulation activity against fungi and erythrocytes, respectively, but rCgGal-2 lacked any agglutinating activity. Carbohydrate binding specificity analysis showed that rCgGal-3 specifically bound D-galactose. Furthermore, rCgGal-2 and rCgGal-3 functioned as opsonin participating in the clearance against invaders in C. gigas. Thus, CgGal-2 with one CRD and CgGal-3 with four CRDs are new members of the galectin family involved in immune responses against bacterial infection. Differences in the organisation and amino acid sequences of CRDs may affect their specificity and affinity for nonself substances.

Identification of Galectin-2-Mucin Interaction and Possible Formation of a High Molecular Weight Lattice.

Galectins comprise a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are proposed to have a protective role in the stomach. As Gal-2 is known to form homodimers in solution, this may result in crosslinking of macromolecules with the sugar structures recognized by Gal-2. In this study, we report that Gal-2 could interact with mucin, an important component of gastric mucosa, in a ß-galactoside-dependent manner. Furthermore, Gal-2 and mucin could form an insoluble precipitate, potentially through the crosslinking of mucins via Gal-2 and the formation of a lattice, resulting in a large insoluble complex. Therefore, we suggest that Gal-2 plays a role in the gastric mucosa by strengthening the barrier structure through crosslinking the mucins on the mucosal surface.

A proto-type galectin-2 from rock bream (Oplegnathus fasciatus): Molecular, genomic, and expression analysis, and recognition of microbial pathogens by recombinant protein.

A ß-galactoside binding lectin, designated as galectin-2, was identified and characterized from rock bream Oplegnathus fasciatus (OfGal-2). The cDNA of OfGal-2 comprised of 692 bp with a coding sequence of 396 bp, encoding a putative polypeptide of 131 amino acids. Gene structure analysis of OfGal-2 revealed a four exon-three intron organization. A single carbohydrate-binding domain containing all seven important residues for carbohydrate binding was located in the third exon, which formed a carbohydrate-binding pocket. Homology screening and sequence analysis demonstrated that OfGal-2 is an evolutionarily conserved proto-type galectin. OfGal-2 transcripts were detected in several healthy fish tissues, with the highest level observed in the intestine, followed by the liver. The expression of OfGal-2 was elevated upon the injection of various mitogenic stimulants and pathogens in a time-dependent manner. Upregulated expression in the liver after tissue injury suggested its role as a damage-associated molecular pattern. Recombinant OfGal-2 protein had hemagglutinating potential and possessed affinity towards lactose and galactose. Moreover, the recombinant protein agglutinated and bound potential pathogenic bacteria and a ciliate. The results of this study indicate that the galectin-2 from rock bream has a potential role in immunity, particularly in the recognition of invading pathogens.

Human galectin-2 interacts with carbohydrates and peptides non-classically: new insight from X-ray crystallography and hemagglutination.

Galectin-2 (Gal-2) plays a role in cancer, myocardial infarction, immune response, and gastrointestinal tract diseases. The only reported crystal structure of Gal-2 shows that it is a dimer in which the monomer subunits have almost identical structures, each binding with one molecule of lactose. In this study, we crystallized Gal-2 under new conditions that produced three crystal structures. In each Gal-2 dimer structure, lactose was shown to be bound to only one of the carbohydrate recognition domain subunits. In solution studies, the thermal shift assay demonstrated that inequivalent monomer subunits in the Gal-2 dimer become equivalent upon ligand binding. In addition, galectin-mediated erythrocyte agglutination assays using lactose and larger complex polysaccharides as inhibitors showed the structural differences between Gal-1 and Gal-2. Overall, our results reveal some novel aspects to the structural differentiation in Gal-2 and expand the potential for different types of molecular interactions that may be specific to this lectin.

Identification of the cysteine residue responsible for oxidative inactivation of mouse galectin-2.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Mouse galectin-2 (mGal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are uniquely sensitive to S-nitrosylation. We have previously reported that oxidation of mGal-2 by hydrogen peroxide (H2O2) resulted in the loss of sugar-binding ability, whereas pre-treatment of mGal-2 with S-nitrosocysteine prevented H2O2-induced inactivation. In this study, we used point-mutated recombinant mGal-2 proteins to study which of the two highly conserved Cys residues in mGal-2 must be S-nitrosylated for protection against oxidative inactivation. Mutation of Cys57 to a Met residue (C57M) did not result in lectin inactivation following H2O2 treatment, whereas Cys75 mutation to Ser (C75S) led to significantly reduced lectin activity, as is the case for wild-type mGal-2. However, pre-treatment of the C75S mutant with S-nitrosocysteine protected the protein from H2O2-induced inactivation. Therefore, Cys57 is suggested to be responsible for oxidative inactivation of the mGal-2 protein, and protection of the sulfhydryl group of the Cys57 in mGal-2 by S-nitrosylation is likely important for maintaining mGal-2 protein function in an oxidative environment such as the gastrointestinal tract.

Immunohistochemical Studies on Galectin Expression in Colectomised Patients with Ulcerative Colitis.

INTRODUCTION: The aetiology and pathogenesis of ulcerative colitis (UC) are essentially unknown. Galectins are carbohydrate-binding lectins involved in a large number of physiological and pathophysiological processes. Little is known about the role of galectins in human UC. In this immunohistochemical exploratory study, both epithelial and inflammatory cell galectin expression were studied in patients with a thoroughly documented clinical history and were correlated with inflammatory activity. MATERIAL AND METHODS: Surgical whole intestinal wall colon specimens from UC patients (n = 22) and controls (n = 10) were studied. Clinical history, pharmacological treatment, and modified Mayo-score were recorded. Tissue inflammation was graded, and sections were stained with antibodies recognizing galectin-1, galectin-2, galectin-3, and galectin-4. RESULTS: Galectin-1 was undetectable in normal and UC colonic epithelium, while galectin-2, galectin-3, and galectin-4 were strongly expressed. A tendency towards diminished epithelial expression with increased inflammatory grade for galectin-2, galectin-3, and galectin-4 was also found. In the inflammatory cells, a strong expression of galectin-2 and a weak expression of galectin-3 were seen. No clear-cut correlation between epithelial galectin expression and severity of the disease was found. CONCLUSION: Galectin expression in patients with UC seems to be more dependent on disease focality and individual variation than on degree of tissue inflammation.

Molecular genetics of coronary artery disease.

Coronary artery disease (CAD) including myocardial infarction (MI) is a common disease and among the leading cause of death in the world. The onset of CAD depends on complex interactions of environmental and genetic factors. To clarify the genetic architecture of MI, we started a genome-wide association study (GWAS) using nearly 100 000 gene-based single-nucleotide polymorphisms (SNPs) from 2000, and identified LTA associated with the increased risk of MI in Japanese population. To our knowledge, this is the first study identified a genetic factor for common disease by GWAS in the worldwide. Through examining the LTA cascade by combination of molecular biological and genetic analyses, we have identified additional MI susceptible genes, LGALS2, PSMA6 and BRAP, so far. Nowadays a lot of large-scale GWAS have identified numerous genetic risk factors for common diseases. In CAD, 51 loci with GWAS significance (P<5 × 10(-8)) have collectively identified by recent large-scale GWAS mainly in Caucasian descent. In this review, we discuss recent advances in molecular genetics for CAD.

Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Polymorphisms of LTA, LGALS2, and PSMA6 genes and coronary atherosclerosis: a pathological study of 1503 consecutive autopsy cases.

OBJECTIVE: Recent genome-wide association studies have identified polymorphisms of lymphotoxin-α (LTA), galectin-2 (LGALS2), and proteasome subunit a type 6 (PSMA6) genes as genetic risk factors for myocardial infarction (MI). However, their effects on coronary atherosclerosis, an intermediate phenotype of MI, remain largely unknown. METHODS: We investigated the correlation between polymorphisms of the LTA, LGALS2, and PSMA6 genes and the severity of pathological coronary stenosis index (CSI) and MI in 1503 consecutive autopsy cases of Japanese elderly patients. RESULTS: The polymorphisms LTA rs1041981 and LGALS2 rs7291467 were associated with CSI with odds ratios of 1.54 (95% CI, 1.17-2.01; AA+CA over CC) and 1.62 (95% CI, 1.11-2.37; TT over CC+CT), respectively. PSMA6 rs1048990 was not associated with CSI. None of the SNPs was associated with MI in our sample. CONCLUSION: Our findings indicate that the LTA and LGALS2 polymorphisms affect the subclinical phenotype of the coronary artery, which predisposes to the incidence of MI.

Lowered expression of galectin-2 is associated with lymph node metastasis in gastric cancer.

BACKGROUND: Lymph node metastasis (LNM) is recognized as an important factor in the progression of tumor malignancy. It is required to discover molecular markers for the prediction of LNM in gastric cancers (GCs). METHODS: An isotope coded affinity tag (ICAT) method and mass spectrometry were used for the quantitative profiling of LNM-related proteins. Western blot analysis of the identified proteins and immunohistochemistry on a tissue microarray comprising 120 GC cases were performed for validation. RESULTS: We identified 151 differentially expressed proteins (DEPs) with an abundance ratio greater than 1.5-fold. The proteins upregulated in LNM-negative GCs were largely populated with proteins related to cell death. Among the DEPs, galectin-2 was further tested because its expression level was significantly higher in LNM-negative GCs (~12-fold, p < 0.0001) and its expression is known to be not ubiquitous but confined to the gastrointestinal tract. Immunohistochemical analysis revealed that low expression of galectin-2 was significantly associated with LNM (p = 0.031) and advanced clinical stage (p = 0.024). The association of low galectin-2 with LNM was found even in early GCs (p = 0.020). CONCLUSION: Our results show that proteomic analysis coupled with immunohistochemistry using tissue microarray is a useful tool for identifying LNM-associated proteins in GC. Also, loss of galectin-2 might play an important role in the aggressiveness of GC.

Lack of association between lymphotoxin-alpha, galectin-2 polymorphisms and coronary artery disease: a meta-analysis.

OBJECTIVE: Previous case-control studies suggested the single nucleotide polymorphisms of lymphotoxin-alpha (LTA) gene and galectin-2 (LGASL2) gene are associated with coronary artery disease and myocardial infarction. However, other studies did not confirm this relationship. The objective was to assess the relationship of LTA gene, LGALS2 gene and coronary artery disease, using a meta-analysis. METHODS: Databases, including PubMed, EMbase, CBM and CNKI, were searched to get the genetic association studies. Data were extracted by two authors and pooled odds ratio (OR) and 95% confidence interval (CI) were calculated. RESULT: The meta-analysis included 20640 (LTA-A252G) and 10552 (LGALS2-C3279T) cases, 15388 (A252G) and 10545 (C3279T) controls. The pooled OR of 252G was 1.02 (95%CI: 0.97-1.07) compared to wild type allele in dominant model, and was 1.00 (95%CI: 0.94-1.07) in recessive model. The pooled OR of 3279T was 0.95 (95%CI: 0.89-1.01) compared to wild type allele in dominant model, and was 0.89 (95%CI: 0.78-1.00) in recessive model. None of the polymorphisms was found to associate with coronary artery disease. CONCLUSION: In present study, the LTA gene and LGALS2-C3279T are not associated with coronary artery disease.

Genetic variation in the lymphotoxin-alpha pathway and the risk of ischemic stroke in European populations.

BACKGROUND AND PURPOSE: Several genes involved in the lymphotoxin-alpha cascade (LTA, LGALS2, and PSMA6) have been linked with the risk of myocardial infarction. Here, we present a comprehensive analysis of these genes in patients with ischemic stroke (IS). METHODS: Twenty-three single nucleotide polymorphisms (SNPs) from LTA, LGALS2, and PSMA6 were genotyped in 601 German IS patients and 736 matched controls. SNPs and haplotypes were tested for association with overall IS, large vessel stroke, and cardioembolic stroke. Significant associations were replicated in an independent sample of 843 IS cases and 933 controls from the UK. RESULTS: Only one SNP (rs1048990 in PSMA6) showed association with overall IS, but this was not replicated in the UK sample. Three SNPs showed significant associations with stroke subtypes (P<0.05), but none of these associations could be replicated in the UK population. CONCLUSIONS: Genetic variation in the lymphotoxin-alpha cascade (LTA, LGALS2, and PSMA6) is not a major risk factor for IS.

Galectin-2 3279TT variant protects against the lymphotoxin-alpha 252GG genotype associated ischaemic stroke.

OBJECTIVE: The galectin-2 protein is presumed to play a regulatory role in the intracellular trafficking of the lymphotoxin-alpha (LTA) cytokine. LTA is a pro-inflammatory factor, its 252GG homozygote variant is considered as a susceptibility factor for arteriosclerosis and cardiovascular diseases. By contrast, the galectin-2-encoding gene LGALS2 3279TT homozygote variant has been demonstrated to exert protection against myocardial infarction by reducing the transcriptional level of galectin-2, thereby leading to a reduced extracellular secretion of LTA. METHODS: In the present study, we examined whether the LGALS2 3279TT homozygote variant alone can influence the prevalence of ischaemic stroke, and whether it can interact somehow with the disadvantageous LTA 252GG homozygote variant. Genetic and clinical data of 385 ischemic stroke patients and 303 stroke and neuroimaging alteration-free controls were analysed. RESULTS: The combination of the LGALS2 3279TT and LTA 252GG homozygote was significantly less frequent in the ischemic stroke group (1.56%) than in the controls (5.94%, p<0.00187; overall stroke group: crude OR: 0.25, 95% CI: 0.1-0.64; adjusted OR: 0.03, 95% CI: 0.025-0.71). CONCLUSIONS: This finding suggests a gene-gene interaction.

Single-site Cys-substituting mutation of human lectin galectin-2: modulating solubility in recombinant production, reducing long-term aggregation, and enabling site-specific monoPEGylation.

The effector capacity of endogenous lectins on cell adhesion/growth prompts studies to turn them into pharmaceutically stable forms. Using human galectin-2 as a proof-of-principle model, we first introduced mutations at the site of one of the two Cys residues, that is, C57A, C57M, and C57S. Only the C57M variant was expressed in bacteria in soluble form in high yield. No notable aggregation of the modified homodimeric lectin occurred during 3 weeks of storage. This mutational process also facilitated the site-directed introduction of poly(ethylene glycol) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one chain per subunit in the homodimer. We note that neither the secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results thus document the feasibility of tailoring a human galectin for enhanced stability to aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as growth regulator and the rate of serum clearance.