Identification of Protein Biomarkers for Differentiating Listeria monocytogenes Genetic Lineage III.

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne illness characterized by septicemia, meningitis, encephalitis, abortions, and occasional death in infants and immunocompromised individuals. L. monocytogenes is composed of four genetic lineages (I, II, III, and IV) and fourteen serotypes. The aim of the current study was to identify proteins that can serve as biomarkers for detection of genetic lineage III strains based on simple antibody-based methods. Liquid chromatography (LC) with electrospray ionization tandem mass spectrometry (ESI MS/MS) followed by bioinformatics and computational analysis were performed on three L. monocytogenes strains (NRRL B-33007, NRRL B-33014, and NRRL B-33077), which were used as reference strains for lineages I, II, and III, respectively. Results from ESI MS/MS revealed 42 unique proteins present in NRRL B-33077 and absent in NRRL B-33007 and NRRL B-33014 strains. BLAST analysis of the 42 proteins against a broader panel of >80 sequenced strains from lineages I and II revealed four proteins [TM2 domain-containing protein (NRRL B-33077_2770), DUF3916 domain-containing protein (NRRL B-33077_1897), DNA adenine methylase (NRRL B-33077_1926), and protein RhsA (NRRL B-33077_1129)] that have no homology with any sequenced strains in lineages I and II. The four genes that encode these proteins were expressed in Escherichia coli strain DE3 and purified. Polyclonal antibodies were prepared against purified recombinant proteins. ELISA using the polyclonal antibodies against 12 L. monocytogenes lineage I, II, and III isolates indicated that TM2 protein and DNA adenine methylase (Dam) detected all lineage III strains with no reaction to lineage I and II strains. In conclusion, two proteins including TM2 protein and Dam are potentially useful biomarkers for detection and differentiation of L. monocytogenes lineage III strains in clinical, environmental, and food processing facilities. Furthermore, these results validate the approach of using a combination of proteomics and bioinformatics to identify useful protein biomarkers.

Selective synthesis of pyridyl pyridones and oxydipyridines by transition-metal-free hydroxylation and arylation of 2-fluoropyridine derivatives.

An efficient protocol for the construction of various pyridyl pyridone and oxydipyridine derivatives through a hydroxylation and arylation tandem reaction of 2-fluoropyridines is reported. Under simple transition-metal-free conditions, the reaction provided a series of products in good to excellent yields, and their structures were confirmed by crystal diffraction analysis. Furthermore, the controlling effect of 6-position substituents on the highly selective synthesis of pyridone and oxydipyridine was studied.

Nickel@Siloxene catalytic nanosheets for high-performance CO2 methanation.

Two-dimensional (2D) materials are of considerable interest for catalyzing the heterogeneous conversion of CO2 to synthetic fuels. In this regard, 2D siloxene nanosheets, have escaped thorough exploration, despite being composed of earth-abundant elements. Herein we demonstrate the remarkable catalytic activity, selectivity, and stability of a nickel@siloxene nanocomposite; it is found that this promising catalytic performance is highly sensitive to the location of the nickel component, being on either the interior or the exterior of adjacent siloxene nanosheets. Control over the location of nickel is achieved by employing the terminal groups of siloxene and varying the solvent used during its nucleation and growth, which ultimately determines the distinct reaction intermediates and pathways for the catalytic CO2 methanation. Significantly, a CO2 methanation rate of 100 mmol gNi-1 h-1 is achieved with over 90% selectivity when nickel resides specifically between the sheets of siloxene.

Electrochemical vicinal aminotrifluoromethylation of alkenes: high regioselective acquisition of ß-trifluoromethylamines.

A novel vicinal aminotrifluoromethylation of alkenes using CF3SO2Na as a trifluoromethyl precursor and acetonitrile as an N-nucleophile has been achieved by an electrooxidative strategy. The present electrochemical protocol achieves efficient and highly regioselective difunctionalization of C[double bond, length as m-dash]C bonds under metal-free and external oxidant-free electrolysis conditions, leading to a series of ß-trifluoromethylamine compounds with good to excellent yields. It is confirmed that the reaction involves free radical processes since CF3 radicals are trapped by scavengers and the ß-trifluoromethylated radical is trapped by BHT, and the deuterium-labeling experiments prove that the oxygen in the product comes from water.

Transposon mutagenesis and identification of mutated genes in growth-delayed Edwardsiella ictaluri.

BACKGROUND: Edwardsiella ictaluri is a Gram-negative facultative intracellular anaerobe and the etiologic agent of enteric septicemia of channel catfish (ESC). To the catfish industry, ESC is a devastating disease due to production losses and treatment costs. Identification of virulence mechanisms of E. ictaluri is critical to developing novel therapeutic approaches for the disease. Here, we report construction of a transposon insertion library and identification of mutated genes in growth-delayed E. ictaluri colonies. We also provide safety and efficacy of transposon insertion mutants in catfish. RESULTS: An E. ictaluri transposon insertion library with 45,000 transposants and saturating 30.92% of the TA locations present in the E. ictaluri genome was constructed. Transposon end mapping of 250 growth-delayed E. ictaluri colonies and bioinformatic analysis of sequences revealed 56 unique E. ictaluri genes interrupted by the MAR2xT7 transposon, which are involved in metabolic and cellular processes and mostly localized in the cytoplasm or cytoplasmic membrane. Of the 56 genes, 30 were associated with bacterial virulence. Safety and vaccine efficacy testing of 19 mutants showed that mutants containing transposon insertions in hypothetical protein (Eis::004), and Fe-S cluster assembly protein (IscX, Eis::039), sulfurtransferase (TusA, Eis::158), and universal stress protein A (UspA, Eis::194) were safe and provided significant protection (p < 0.05) against wild-type E. ictaluri. CONCLUSIONS: The results indicate that random transposon mutagenesis causing growth-delayed phenotype results in identification bacterial virulence genes, and attenuated strains with transposon interrupted virulence genes could be used as vaccine to activate fish immune system.

Ferric hydroxamate uptake system contributes to Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in fish, particularly in channel catfish. Ferric iron is an essential micronutrient for bacterial survival, and some bacterial pathogens use secreted hydroxamate-type siderophores to chelate iron in host tissues. Siderophore-iron complexes are taken up by these bacteria via the ferric hydroxamate uptake (Fhu) system. In E. ictaluri, the Fhu system consists of fhuC, fhuD, fhuB, and fhuA genes. However, the importance of the Fhu system in E. ictaluri virulence has not been investigated completely. Here, we present construction of E. ictaluri fhuD and fhuB mutants (EiΔfhuD and EiΔfhuB) by in-frame gene deletion and evaluation of the mutants' virulence and immunogenicity in channel catfish fingerlings and fry. Immersion challenges showed that EiΔfhuD was not significantly attenuated (p < 0.05) in catfish fingerlings, whereas EiΔfhuB was significantly attenuated (p < 0.01). Catfish fingerlings immunized with EiΔfhuD and EiΔfhuB showed 100% and 97.62% survival, respectively. Fry immersion challenges indicated EiΔfhuB was also significantly attenuated (p < 0.05) in two-week old fry compared to the wild-type (48.96% vs. 82.14% mortalities). The survival rate in the fry vaccinated with EiΔfhuB was significantly higher (p < 0.05) than that of non-vaccinated fry (96.77% vs. 21.42% survival). Our data indicates that the fhuB gene, but not the fhuD gene, contributes to E. ictaluri virulence.

Involvement of tolQ and tolR genes in Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative intracellular facultative pathogen causing enteric septicemia of channel catfish (ESC). The Tol system, consisting of four envelope proteins TolQ, TolR, TolA, and TolB, are required for colicin import and contributes to bacterial virulence in several pathogenic bacteria. However, the Tol system and its importance in E. ictaluri virulence have not been investigated. Here we present construction and evaluation of the E. ictaluri TolQ, TolR and TolQR mutants (EiΔtolQ, EiΔtolR, and EiΔtolQR). The Tol mutants were developed using in-frame gene deletion and their attenuation and vaccine efficacy were determined in catfish fingerlings. The EiΔtolQ, EiΔtolR, and EiΔtolQR mutants showed reduced virulence in catfish (28.93%, 19.70%, and 39.82% mortality, respectively) compared to wild type (46.91% mortality). Further, vaccination with these mutants protected catfish against subsequent wild-type infection. This study suggests that the Tol system contributes to E. ictaluri virulence in catfish.

Tricarboxylic acid cycle and one-carbon metabolism pathways are important in Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.

Construction and evaluation of an Edwardsiella ictaluri fhuC mutant.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in channel catfish. Iron is an essential micronutrient needed for bacterial virulence, and to acquire iron, many Gram-negative bacteria secrete ferric iron chelating siderophores. The ferric hydroxamate uptake (Fhu) system consists of four genes (fhuC, fhuD, fhuB, and fhuA), and is involved in the uptake of hydroxamate type siderophores across bacterial membranes. However, the Fhu system and its importance in E. ictaluri virulence have been uninvestigated. Here, we present construction and evaluation of an E. ictaluri ΔfhuC mutant. The E. ictaluri fhuC gene was deleted in-frame by allelic exchange, and the mutant's growth in media and virulence in catfish were determined. Our results indicated that deletion of the E. ictaluri fhuC gene did not affect the growth of E. ictaluri largely in both iron-replete and iron-depleted media. Addition of ferric iron sources into the iron-depleted medium improved the growth of both E. ictaluri ΔfhuC and wild type (WT). Catfish mortalities indicated that E. ictaluri ΔfhuC mutant was attenuated 2.05-fold compared with the parent strain. The catfish immunized with the E. ictaluri ΔfhuC mutant showed a high relative percent survival rate (97.50%) after re-challenge with the WT E. ictaluri strain. Taken together, our data indicates that the fhuC gene contributes to E. ictaluri virulence.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and cardiac fibroblast growth.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) regulates growth of a variety of cells and is important in inflammation, oxidative stress, and tissue remodeling. Recent studies show that LOX-1 deletion limits cardiac remodeling after sustained hypertension. We posited that LOX-1 may affect cardiac fibroblast growth and collagen secretion. To examine this postulate, we studied growth pattern of cardiac fibroblasts from hearts of wild-type and LOX-1 knockout (KO) mice. LOX-1 KO fibroblasts exhibited dramatically reduced growth when compared with wild-type mice fibroblasts and became much larger than wild-type mice fibroblasts in serial cultures, suggesting arrest of cell division. Western blotting and immunofluorescence showed that cell division control protein 42, a key regulator for cell division, was markedly downregulated in LOX-1 KO fibroblasts. The cytoskeletal organization in these fibroblasts was significantly altered in strand orientation, and some fibroblasts were completely devoid of F-actin. Furthermore, NADPH oxidase expression and generation of reactive oxygen species, as well as cell proliferation signals serine/threonine-specific protein kinase and murine double minute 2, were significantly reduced in LOX-1 KO fibroblasts. To confirm the essential role of LOX-1 in fibroblast growth, LOX-1 KO fibroblasts were transfected with h-LOX-1 cDNA. After transfection, the altered pattern of cytoskeletal organization, as well as expression of cell division control protein 42, serine/threonine-specific protein kinase, and murine double minute 2, was normalized. In congruent with these in vitro data, we found that the cardiac fibroblast number and expression of fibronectin and procoallagen-1/collagen were significantly lower in hypertensive LOX-1 KO mice hearts than in hypertensive wild-type mice hearts subjected to sustained hypertension (angiotensin II infusion). These findings implicate LOX-1 in cytoskeletal organization and growth of cardiac fibroblasts.

Abrogation of lectin-like oxidized LDL receptor-1 attenuates acute myocardial ischemia-induced renal dysfunction by modulating systemic and local inflammation.

It is assumed that acute myocardial infarction affects renal function. To study the mechanism, we used mice following permanent ligation of their left coronary artery that results in extensive myocardial infarction. Soon after ligation, there was a marked rise in circulating pro-inflammatory cytokines and malondialdehyde (thiobarbituric acid-positive evidence of lipid peroxidation). Renal function had significantly declined by the third day in association with mild fibrosis, and swelling of glomeruli and tubules. There was a significant increase in the expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), interelukin-1ß, vascular cell adhesion molecule-1, and thiobarbituric acid-reactive substances in the kidney. Renal function showed some recovery by Day 21; however, there was progressive fibrosis of the kidneys. LOX-1 knockout mice had significantly diminished increases in systemic and renal pro-inflammatory cytokines, malondialdehyde, structural alterations, and decline in renal function than the wild-type mice following ligation of the left coronary artery. Cardiac function and survival rates were also significantly better in the LOX-1 knockout mice than in the wild-type mice. Hence, severe myocardial ischemia results in renal dysfunction and histological abnormalities suggestive of acute renal injury. Thus, LOX-1 is a key modulator among multiple mechanisms underlying renal dysfunction following extensive myocardial infarction.

Delineation of the effects of angiotensin type 1 and 2 receptors on HL-1 cardiomyocyte apoptosis.

Angiotensin II (Ang II) exerts its effects by activating its receptors, primarily type 1 (AT1R) and type 2 (AT2R). While the role of AT1R activation in cardiomyocyte physiology is well known, the role of AT2R in cardiomyocyte apoptosis remains controversial. To define the precise role of AT1R and AT2R in this process, we transfected HL-1 cardiomyocytes with AT1R or AT2R cDNA, and examined markers of apoptosis. We found that AT1R overexpression was associated with upregulation of endogenous AT2R expression, but AT2R overexpression did not affect endogenous AT1R expression. Caspase-3 staining indicated that overexpression of AT1R as well as AT2R resulted in cardiomyocyte apoptosis with appropriate alterations in annexin V, Bax and Bcl2 expression. Overexpression of AT1R and AT2R markedly increased IL-1ß (AT2R>AT1R), iNOS (AT2R>AT1R) and eNOS expression. AT2R-induced cell apoptosis could be blocked by the iNOS selective inhibitor 1,400 W, and did not require exogenous Ang II. These findings suggest that AT2R overexpression induces cardiomyocyte apoptosis, most likely via iNOS upregulation. AT1R-mediated cardiomyocyte apoptosis may be partially mediated by upregulation of endogenous AT2R.

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription.

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22(phox), p47(phox), p67(phox), NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H2O2. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety.

Cross-talk between inflammation and angiotensin II: studies based on direct transfection of cardiomyocytes with AT1R and AT2R cDNA.

Ischemic myocardium exhibits inflammation, local angiotensin II (Ang II) generation and up-regulation of LOX-1, a lectin-like ox-LDL receptor. To define the inter-active roles of Ang II and inflammation in furthering tissue injury, cultured HL-1 cardiomyocytes were treated with Ang II. Ang II treatment up-regulated the expression of Ang II type 1 (AT1R) and type 2 (AT2R) receptors as well as LOX-1. Ang II also activated p44/42MAPK, p38MAPK, c-Jun and NF-κB, and increased the expression of inflammation-related genes (interleukins-6, interleukins-10, tumor necrosis factor-α, intercellular adhesion molecule-1). To study how inflammation per se might affect expression of Ang II receptors and LOX-1, cultured, cardiomyocytes were treated with lipopolysaccharide (LPS). Like Ang II, LPS increased the expression of AT1R, AT2R and LOX-1. LPS also activated mitogen-acticated protein kinase (MAPKs), c-Jun and NF-κB, and pro-inflammatory genes. The selective inhibitors of MAPKs, c-Jun and NF-κB each blocked the transcription of LOX-1 and pro-inflammatory genes in response to Ang II as well as LPS. These observations suggested a positive feedback between Ang II and inflammation. To delineate the role of AT1R and AT2R in LOX-1 expression, another set of cardiomyocytes were transfected with AT1R or AT2R cDNA. Forced over-expression of AT1R resulted in activation of MAPKs, c-Jun and NF-κB, up-regulation of inflammatory genes and LOX-1; on the other hand forced AT2R over-expression induced up-regulation of pro-apoptotic signals (pro-IL-1ß and IL-1ß), and decreased LOX-1 expression. These studies show that both Ang II and inflammation mediator LPS up-regulate AT1R, AT2R and LOX-1 expression. Up-regulation of AT1R promotes inflammation and LOX-1 expression, whereas up-regulation of AT2R promotes apoptosis signals and decreases LOX-1 expression.

Insights into high affinity small ubiquitin-like modifier (SUMO) recognition by SUMO-interacting motifs (SIMs) revealed by a combination of NMR and peptide array analysis.

The small ubiquitin-like modifiers (SUMOs) regulate many essential cellular functions. Only one type of SUMO-interacting motif (SIM) has been identified that can extend the ß-sheet of SUMO as either a parallel or an antiparallel strand. The molecular determinants of the bound orientation and paralogue specificity of a SIM are unclear. To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. In addition, the SIM sequence requirements have been investigated by peptide arrays in comparison with another high affinity SIM that binds in the opposing orientation. We found that antiparallel binding SIMs tolerate more diverse sequences, whereas the parallel binding SIMs prefer the more strict sequences consisting of (I/V)DLT that have a preference in high affinity SUMO2 and -3 binding. Comparison of two high affinity SUMO1-binding SIMs that bind in opposing orientations has revealed common SUMO1-specific interactions needed for high affinity binding. This study has significantly advanced our understanding of the molecular determinants underlining SUMO-SIM recognition.

LOX-1: a critical player in the genesis and progression of myocardial ischemia.

Myocardial ischemia is the most common cause of mortality and morbidity in the developed countries and rapidly becoming a common malady in the developing countries. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the genesis and progression of atherosclerosis and its complications. LOX-1 has been identified as a major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, cardiomyocytes and fibroblast. In vitro and in vivo studies show that LOX-1 is upregulated during acute myocardial ischemia, and continues to be upregulated during chronic ischemia. Further, LOX-1 inhibition reduces ischemic myocardial injury and limits cardiac remodeling. LOX-1 inhibition decreases oxidative stress and inflammatory response to injury resulting in limitation of ischemic injury. Molecular studies show that LOX-1 inhibition reduces release of pro-inflammatory cytokines and expression of angiotensin II type 1 receptor via inhibition of redox-sensitive pathways. These alterations limit cardiomyocyte hypertrophy and collagen accumulation in the ischemic regions. These alterations in molecular signaling and physical alterations can result in improved cardiac function and better survival after ischemic myocardial injury.

Oxidized low-density lipoprotein and atherosclerosis implications in antioxidant therapy.

Low-density lipoprotein (LDL)-cholesterol is important for cellular function, but in high concentrations, it can lead to atheroma formation. Over the past several decades, it has become abundantly evident that the oxidized form of LDL-cholesterol (ox-LDL) is more important in the genesis and progression of atherosclerosis than native unmodified LDL-cholesterol. Ox-LDL leads to endothelial dysfunction, an initial step in the formation of an atheroma. Ox-LDL acts via binding to a number of scavenger receptors (SR), such as SR-A1, SR-A2 and lectin-like oxidized low-density lipoprotein receptor (LOX-1). Ox-LDL can upregulate expression of its own receptor LOX-1 on endothelial cells and activate these cells. In addition, ox-LDL promotes the growth and migration of smooth muscle cells, monocytes/macrophages and fibroblasts. Ox-LDL also leads to the generation of reactive oxygen species that in physiologic concentrations combat invasion of the body by noxious agents, but when in excess, can lead to a state of oxidative stress. There is evidence for the presence of oxidative stress in a host of conditions such as atherosclerosis and aging. In this review, we discuss the role of oxidative stress, ox-LDL and LOX-1 in atherogenesis and the reasons why the traditional approaches to limit oxidant stress have not been successful.

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation.

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.

Protective effect of caffeine administration on myocardial ischemia/reperfusion injury in rats.

Many studies have examined the association between coffee consumption and risk of cardiovascular disease, but the results remain controversial. Caffeine is one of the main biologically active compounds of coffee. The aim of this study was to investigate the potential role of caffeine on myocardial ischemia/reperfusion (I/R) injury in the rats. We administered caffeine (25 mg/kg per day) or saline in rats for 4 weeks before myocardial ischemia/reperfusion operation. Compared with the sham group, caffeine treatment decreased ischemia-associated infarct size, serum creatine kinase, and lactate dehydrogenase 3-h reperfusion after 30-min ischemia. Myocardial neutrophil infiltration (assessed by myeloperoxidase activity) was significantly decreased compared with the control group. Meanwhile, caffeine reduced the myocardial apoptosis and suppressed the activation of caspase 3 during myocardial I/R. Importantly, we observed a strong poly(ADP-ribose) polymerase (PARP) activation during myocardial I/R, and caffeine administration inhibited PARP activation and attenuated the expression of PARP-related proinflammatory mediators such as inducible nitric oxide synthetase, IL-6, and TNF-α, all of which may be correlated with downregulated nuclear factor κB activity. We concluded that caffeine protected against myocardial I/R injury by inhibiting inflammation and apoptosis.

Oxidative stress and lectin-like ox-LDL-receptor LOX-1 in atherogenesis and tumorigenesis.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been identified as a major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, monocytes, platelets, cardiomyocytes, and vascular smooth muscle cells. Its expression is minimal under physiological conditions but can be induced under pathological conditions. The upregulation of LOX-1 by ox-LDL appears to be important for physiologic processes, such as endothelial cell proliferation, apoptosis, and endothelium remodeling. Pathophysiologic effects of ox-LDL in atherogenesis have also been firmly established, including endothelial cell dysfunction, smooth muscle cell growth and migration, monocyte transformation into macrophages, and finally platelet aggregation-seen in atherogenesis. Recent studies show a positive correlation between increased serum ox-LDL levels and an increased risk of colon, breast, and ovarian cancer. As in atherosclerosis, ox-LDL and its receptor LOX-1 activate the inflammatory pathway through nuclear factor-kappa B, leading to cell transformation. LOX-1 is important for maintaining the transformed state in developmentally diverse cancer cell lines and for tumor growth, suggesting a molecular connection between atherogenesis and tumorigenesis.