Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.     

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.     

Regulation of the Werner helicase through a direct interaction with a subunit of protein kinase A

Advanced glycation end products (AGE) are known to serve as ligands for the scavenger receptors such as SR-A, CD36 and SR-BI. In the current study, we examined whether AGE is recognized by lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Cellular binding experiments revealed that AGE-bovine serum albumin (AGE-BSA) showed the specific binding to CHO cells overexpressing bovine LOX-1 (BLOX-1), which was effectively suppressed by an anti-BLOX-1 antibody. Cultured bovine aortic endothelial cells also showed the specific binding for AGE-BSA, which was suppressed by 67% by the anti-BLOX-1 antibody. Thus, LOX-1 is identified as a novel endothelial receptor for AGE.     

Structural variation and inhibitor binding in polypeptide deformylase from four different bacterial species

Polypeptide deformylase (PDF) catalyzes the deformylation of polypeptide chains in bacteria. It is essential for bacterial cell viability and is a potential antibacterial drug target. Here, we report the crystal structures of polypeptide deformylase from four different species of bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Comparison of these four structures reveals significant overall differences between the two Gram-negative species (E. coli and H. influenzae) and the two Gram-positive species (S. pneumoniae and S. aureus). Despite these differences and low overall sequence identity, the S1' pocket of PDF is well conserved among the four enzymes studied. We also describe the binding of nonpeptidic inhibitor molecules SB-485345, SB-543668, and SB-505684 to both S. pneumoniae and E. coli PDF. Comparison of these structures shows similar binding interactions with both Gram-negative and Gram-positive species. Understanding the similarities and subtle differences in active site structure between species will help to design broad-spectrum polypeptide deformylase inhibitor molecules.     

Curcumin inhibits TNFα-induced lectin-like oxidised LDL receptor-1 (LOX-1) expression and suppresses the inflammatory response in human umbilical vein endothelial cells (HUVECs) by an antioxidant mechanism

In this study, the anti-oxidative activities of 70% ethanol extract from Curcuma aromatica Salisb. (CAS) and curcumin (CUR) were studied. The CAS extracts and CUR were both found to have a potent scavenging activity against the reactive species tested, as well as an inhibitory effect on LDL oxidation. Cultured human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor α (TNFα), expression of intracellular reactive oxygen species (ROS), nitric oxide (NO), endothelial nitric oxide synthase (eNOS), lectin-like oxidised LDL receptor-1 (LOX-1), adhesion molecules, inhibitory kappa Bα (IκBα) and nuclear factor kappa B (NFκB) were measured. In HUVECs stimulated with TNFα, CUR significantly suppressed expression of the intracellular ROS, LOX-1 and adhesion molecules, degradation of IκBα and translocation of NFκB, while inducing production of NO by phosphorylation of eNOS (p <0.05). In conclusion, CAS and CUR may modulate lipoprotein composition and attenuate oxidative stress by elevated antioxidant processes.     

LOX-1 expressed in cultured rat chondrocytes mediates oxidized LDL-induced cell death-possible role of dephosphorylation of Akt

The oxidative changes of lipids in cartilage proceed with ageing and with the grade of osteoarthritis. To clarify the role of oxidatively modified lipids in articular cartilage in osteoarthritis, here, we investigated lectin-like oxidized LDL receptor (LOX-1) in rat cultured articular chondrocytes. LOX-1 expression was detectable in basal culture condition and enhanced by the treatment of oxidized LDL and interleukin-1beta. DiI-labeled oxidized LDL was bound and ingested by chondrocytes via LOX-1. Oxidized LDL dose-dependently reduced chondrocyte viability, inducing non-apoptotic cell death, which was again suppressed by anti-LOX-1 antibody treatment. Oxidized LDL reduced the amount of phosphorylated Akt, a substrate of PI3 kinase via LOX-1. Consistently, the PI3 kinase inhibitor, LY294002, decreased cell viability dose-dependently, and the PI3 kinase activator, IGF-I, reversed the effect of oxidized LDL on the cell death. LOX-1 might be involved in the pathogenesis of osteoarthritis, inducing chondrocyte death through PI3 kinase/Akt pathway.     

Cutting edge: SR-PSOX/CXC chemokine ligand 16 mediates bacterial phagocytosis by APCs through its chemokine domain

SR-PSOX and CXC chemokine ligand (CXCL)16, which were originally identified as a scavenger receptor and a transmembrane-type chemokine, respectively, are indicated to be identical. In this study, we demonstrate that membrane-bound SR-PSOX/CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria. Importantly, our prepared anti-SR-PSOX mAb, which suppressed chemotactic activity of SR-PSOX, significantly inhibited bacterial phagocytosis by human APCs including dendritic cells. Various scavenger receptor ligands inhibited the bacterial phagocytosis of SR-PSOX. In addition, the recognition specificity for bacteria was determined by only the chemokine domain of SR-PSOX/CXCL16. Thus, SR-PSOX/CXCL16 may play an important role in facilitating uptake of various pathogens and chemotaxis of T and NKT cells by APCs through its chemokine domain.     

LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation

A recently identified lectin-like oxidized low-density lipoprotein receptor (LOX-1) mediates endothelial cell injury and facilitates inflammatory cell adhesion. We studied the role of LOX-1 in myocardial ischemia-reperfusion (I/R) injury. Anesthetized Sprague-Dawley rats were subjected to 60 min of left coronary artery (LCA) ligation, followed by 60 min of reperfusion. Rats were treated with saline, LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat IgG (10 mg/kg) before I/R. Ten other rats underwent surgery without LCA ligation and served as a sham control group. LOX-1 expression was markedly increased during I/R (P < 0.01 vs. sham control group). Simultaneously, the expression of matrix metalloproteinase-1 (MMP-1) and adhesion molecules (P-selectin, VCAM-1, and ICAM-1) was also increased in the I/R area (P < 0.01 vs. sham control group). There was intense leukocyte accumulation in the I/R area in the saline-treated group. Treatment of rats with the LOX-1 antibody prevented I/R-induced upregulation of LOX-1 and reduced MMP-1 and adhesion molecule expression as well as leukocyte recruitment. LOX-1 antibody, but not nonspecific IgG, also reduced myocardial infarct size (P < 0.01 vs. saline-treated I/R group). To explore the link between LOX-1 and adhesion molecule expression, we measured expression of oxidative stress-sensitive p38 mitogen-activated protein kinase (p38 MAPK). The activity of p38 MAPK was increased during I/R (P < 0.01 vs. sham control), and use of LOX-1 antibody inhibited p38 MAPK activation (P < 0.01). These findings indicate that myocardial I/R upregulates LOX-1 expression, which through p38 MAPK activation increases the expression of MMP-1 and adhesion molecules. Inhibition of LOX-1 exerts an important protective effect against myocardial I/R injury.     

Conserved interactions in the Staphylococcus aureus DNA PolC chromosome replication machine

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, tau, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli tau. S. aureus tau also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III.tau. We demonstrate that the extreme C-terminal residues of PolC bind to and function with beta clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus delta wrench of the clamp loader binds to E. coli beta. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes beta clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous beta clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.     

Differential regulation of CCL-11/eotaxin-1 and CXCL-8/IL-8 by Gram-positive and Gram-negative bacteria in human airway smooth muscle cells

Background

Bacterial infections are a cause of exacerbation of airway disease. Airway smooth muscle cells (ASMC) are a source of inflammatory cytokines/chemokines that may propagate local airway inflammatory responses. We hypothesize that bacteria and bacterial products could induce cytokine/chemokine release from ASMC.

Methods

Human ASMC were grown in culture and treated with whole bacteria or pathogen associated molecular patterns (PAMPs) for 24 or 48 h. The release of eotaxin-1, CXCL-8 or GMCSF was measured by ELISA.

Results

Gram-negative E. coli or Gram-positive S. aureus increased the release of CXCL-8, as did IL-1β, LPS, FSL-1 and Pam₃CSK4, whereas FK565, MODLys18 or Poly I:C did not. E. coli inhibited eotaxin-1 release under control conditions and after stimulation with IL-1β. S. aureus tended to inhibit eotaxin-1 release stimulated with IL-1β. E. coli or LPS, but not S. aureus, induced the release of GMCSF.

Conclusion

Gram-positive or Gram-negative bacteria activate human ASMC to release CXCL-8. By contrast Gram-negative bacteria inhibited the release of eotaxin-1 from human ASMCs. E. coli, but not S. aureus induced GMCSF release from cells.Our findings that ASMC can respond directly to Gram-negative and Gram-positive bacteria by releasing the neutrophil selective chemokine, CXCL-8, is consistent with what we know about the role of neutrophil recruitment in bacterial infections in the lung. Our findings that bacteria inhibit the release of the eosinophil selective chemokine, eotaxin-1 may help to explain the mechanisms by which bacterial immunotherapy reduces allergic inflammation in the lung.     

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 μg ml(-1) (FA) and 5000 μg ml(-1) (GA), for L. monocytogenes it was >5000 μg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 μg ml(-1) (FA) and >5000 μg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.     

Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.     

Lipid peroxidation modification of protein generates Nepsilon-(4-oxononanoyl)lysine as a pro-inflammatory ligand

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.     

Non-woven fibrous materials with antibacterial properties prepared by tailored attachment of quaternized chitosan to electrospun mats from maleic anhydride copolymer

In order to impart antibacterial properties to microfibrous electrospun materials from styrene/maleic anhydride copolymers, quaternized chitosan derivatives (QCh) containing alkyl substituents of different chain lengths are covalently attached to the mats. A complete inhibition of the growth of bacteria, S. aureus (Gram-positive) and E. coli (Gram-negative), for a contact time of 30–120 min or a decrease of the bacterial titer by 2–3 log units is observed depending on the quaternization degree, the chain length of the alkyl substituent, and the molar mass of QCh. The modified mats are also effective in suppressing the adhesion of pathogenic S. aureus bacteria.     

Endothelial scavenger receptors

In the past few decades, cDNAs for endothelial scavenger receptors that bind to negatively charged molecules, particularly acetylated low density lipoproteins (Ac-LDL), have been cloned by expression cloning using modified LDL as ligands. A prototypic members of endothelial scavenger receptor family, namely, scavenger receptor class B type I (SR-BI) has been characterized as a high density lipoprotein (HDL) receptor. Another prototypic member, CD36, has been determined as a multiple ligand receptor because it binds to oxidized LDLs (Ox-LDL), trombospondin, erythrocytes infected with Plasmodium falciparum, long-chain fatty acids, and Gram-negative and Gram-positive bacteria. Lectin-like oxidized LDL receptor-1 (LOX-1) has been discovered as the principal receptor that mediates the action of Ox-LDL in the vascular walls. Recently, the structure of oxidized phospholipids, originally found in Ox-LDL, and its molecular mechanism of action on endothelial cells were determined. Further, the use of genetically manipulated rodent models and the recent forward genetic screening technique revealed the physiological and pathological functions of these endothelial scavenger receptors in innate immunity and infection. In this review, the structure and function of these multiligand scavenger receptors of endothelial cells have been described mainly in relation with lipid metabolism.     

LOX-1 Plays an Important Role in Ischemia-Induced Angiogenesis of Limbs

LOX-1, lectin-like oxidized low-density lipoprotein (LDL) receptor-1, is a single transmembrane receptor mainly expressed on endothelial cells. LOX-1 mediates the uptake of oxidized LDL, an early step in atherosclerosis; however, little is known about whether LOX-1 is involved in angiogenesis during tissue ischemia. Therefore, we examined the role of LOX-1 in ischemia-induced angiogenesis in the hindlimbs of LOX-1 knockout (KO) mice. Angiogenesis was evaluated in a surgically induced hindlimb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD) and arteriole density (AD). After right hindlimb ischemia, the ischemic/nonischemic hindlimb blood flow ratio was persistently lower in LOX-1 KO mice than in wild-type (WT) mice. CD and AD were significantly smaller in LOX-1 KO mice than in WT mice on postoperative day 14. Immunohistochemical analysis revealed that the number of macrophages infiltrating ischemic tissues was significantly smaller in LOX-1 KO mice than in WT mice. The number of infiltrated macrophages expressing VEGF was also significantly smaller in LOX-1 KO mice than in WT mice. Western blot analysis and ROS production assay revealed that LOX- KO mice show significant decrease in Nox2 expression, ROS production and HIF-1α expression, the phosphorylation of p38 MAPK and NF-κB p65 subunit as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and LOX-1 itself in ischemic muscles, which is supposed to be required for macrophage infiltration expressing angiogenic factor VEGF. Reduction of VEGF expression successively suppressed the phosphorylation of Akt and eNOS, which accelerated angiogenesis, in the ischemic leg of LOX-1 KO mice. Our findings indicate that LOX-1 plays an important role in ischemia-induced angiogenesis by 1) Nox2-ROS-NF-κB activation, 2) upregulated expression of adhesion molecules: VCAM-1 and LOX-1 and 3) promoting macrophage infiltration, which expresses angiogenic factor VEGF.     

Granulosa cell subtypes respond by autophagy or cell death to oxLDL-dependent activation of the oxidized lipoprotein receptor 1 and toll-like 4 receptor

Autophagic cell death has been observed in granulosa cell cultures via the oxLDL-dependent activation of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1). This activation might differ for cytokeratin-positive (CK⁺) and CK^- granulosa cells. In particular, LOX-1 and Toll-like receptor 4 (TLR4), one of the pattern recognition receptors of innate immunity, might be diversely regulated. Granulosa cell subtype cultures were established from the follicle harvests of patients undergoing in vitro fertilization (IVF) therapy. In response to oxLDL treatment, the fibroblast-like CK- cells upregulated LOX-1 and exhibited reparative autophagy, which could be blocked with anti-LOX-1 antibody. The epithelioid-like CK⁺ cells did not regulate LOX-1 expression upon oxLDL application, but the expression of TLR4 and CD14 increased between 0 and 36 h of oxLDL/nDL treatment. This up-regulation was associated with non-apoptotic cell death based on the absence of cleaved caspase-3. Reactive oxygen species (ROS) increased with 12 h oxLDL application and steroidogenic acute regulatory (StAR) protein expression was negligible. In CK^- cells, the inhibition of TLR4 downregulated LOX-1 and induced apoptosis. We concluded that CK- granulosa cells are protected against oxLDL-dependent apoptosis by TLR4, whereas, in CK⁺ cells, oxLDL-induced TLR4 activation triggers non-apoptotic cell death. The CK⁺ cells might represent immune-like granulosa cells involved in ovarian remodeling processes.     

Cadmium uptake by growing cells of gram-positive and gram-negative bacteria

The present study evaluates the effect of the cadmium (Cd2+) on the growth and protein synthesis of some Gram-positive (Staphylococcus aureus, Bacillus subtilis and Streptococcus faecium) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and the cadmium uptake by the same micro-organisms. The Gram-negative bacteria tested were less sensitive to metal ions than the Gram-positive, and P. aeruginosa was the most resistant. The Gram-negative bacteria were also able to accumulate higher amounts of cadmium during growth than the Gram-positive bacteria. The maximum values of specific metal uptake (microgram of Cd2+ incorporated per mg of protein) were: 0.52 for S. aureus, 0.65 for S. faecium, 0.79 for B. subtilis, 2.79 for E. coli and 24.15 for P. aeruginosa, respectively. The differences in the ability to accumulate metal found between Gram-negative and Gram-positive bacteria seems to account for different mechanisms of metal resistance.