Shan-Rui Han

Possible Protective Role for C-Reactive Protein in Atherogenesis Complement Activation by Modified Lipoproteins Halts Before Detrimental Terminal Sequence

Background—Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)–dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion. Methods and Results—E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase. Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated complement to completion, independent of CRP, when present above a threshold concentration (100 to 200 μg/mL in 5% serum). Below the threshold, all E-LDL preparations activated complement in dependence of CRP, but the pathway then halted before the terminal sequence. Native LDL and oxidized LDL did not activate complement under any circumstances tested. Immunohistological analyses corroborated the concept that CRP-dependent complement activation inefficiently generates C5b-9. Conclusions—Binding of CRP to E-LDL is the first trigger for complement activation in the atherosclerotic lesion, but the terminal sequence is thereby spared. This putatively protective function of CRP is overrun at higher E-LDL concentrations, so that potentially harmful C5b-9 complexes are generated.

Beyond cholesterol: the enigma of atherosclerosis revisited.

Atherosclerosis is widely regarded as a chronic inflammatory disease that develops as a consequence of entrapment of low density lipoprotein (LDL) in the arterial intima. Native LDL lacks inflammatory properties, so the lipoprotein must undergo biochemical alterations in order to become atherogenic. Modification is commonly regarded as being dangerous because it bestows inflammatory properties onto the lipoprotein. Most current models consider oxidation to be the decisive modifying event. Here, we submit a different concept for discussion. We propose that modification of tissue-entrapped LDL is required because it enables the lipoprotein to signal to the immune system and effect its own removal. Oxidation would be too haphazard to fulfill this function. We summarize the evidence indicating that modification occurs through the action of ubiquitous hydrolytic enzymes. Enzymatically remodeled LDL binds C-reactive protein. C-reactive protein bound to remodeled LDL not only activates complement but also regulates it by inhibiting activation of the terminal complement cascade. Simultaneously, epitopes are exposed to enable the lipoprotein to be recognized and taken up by macrophages. The high density lipoprotein-dependent reverse transport pathway concludes the sequence of events that clear tissues of cholesterol in a non-inflammatory manner very similar to what has been described for the removal of apoptotic cells. It is proposed that these physiological processes occur throughout life without harm, pathology evolving only when the machinery suffers overload. Detrimental effects are then evoked primarily by the unreigned activation of complement, macrophages, and other effectors of the immune system in the lesions.

Pore-forming Staphylococcus aureus α-toxin triggers epidermal growth factor receptor-dependent proliferation

Staphylococcal α‐toxin is an archetypal killer protein that homo‐oligomerizes in target cells to create small transmembrane pores. The membrane‐perforating β‐barrel motif is a conserved attack element of cytolysins of Gram‐positive and Gram‐negative bacteria. Following the recognition that nucleated cells can survive membrane permeabilization, a profile of abundant transcripts was obtained in transiently perforated keratinocytes. Several immediate early genes were found to be upregulated, reminiscent of the cellular response to growth factors. Cell cycle analyses revealed doubling of S + G2/M phase cells 26 h post toxin treatment. Determination of cell counts uncovered that after an initial drop, numbers increased to exceed the controls after 2 days. A non‐lytic α‐toxin mutant remained without effect. The α‐toxin pore is too small to allow egress of cytosolic growth factors, and evidence was instead obtained for growth signalling via the epidermal growth factor receptor (EGFR). Inhibition of the EGFR or of EGFR‐proligand‐processing blocked the mitogenic effect of α‐toxin. Western blots with phospho‐specific antibodies revealed activation of the EGFR, and of the adapter protein Shc. Immediate early response and proliferation upon transient plasma membrane pore formation by bacterial toxins may represent a novel facet of the complex interaction between pathogen and host.

Enzymatic Modification of Low-Density Lipoprotein in the Arterial Wall: A New Role for Plasmin and Matrix Metalloproteinases in Atherogenesis

Objective—Functionally interactive proteases of the plasminogen/plasmin and the matrix metalloproteinase (MMP) system degrade and reorganize the extracellular matrix of the vessel wall in atherosclerosis. Here we investigated whether such proteases are able to confer atherogenic properties onto low density lipoprotein by nonoxidative modification. Methods and Results—Similar to the recently described enzymatically-modified low-density lipoprotein (E-LDL), native LDL exposed to plasmin or matrix MMP-2 or MMP-9 and cholesterylester-hydrolase (CEH) showed extensive deesterification, with ratios of free cholesterol to total cholesterol rising to 0.8 compared with 0.2 in native LDL. When the ratio exceeded 0.6, both plasmin/CEH-LDL and MMP/CEH-LDL fused into larger particles. In parallel, they gained C-reactive protein–dependent complement-activating capacity. E-LDL produced with any protease/CEH combination was efficiently taken up by human macrophages, whereby marked induction of MMP-2 expression by E-LDL was observed. These in vitro findings had their in vivo correlates: urokinase-type plasminogen activator, MMP-2, and MMP-9 were detectable in both early and advanced human atherosclerotic lesions in colocalization with E-LDL. Conclusions—Plasmin and MMP-2/MMP-9 may not only be involved in remodeling of the extracellular matrix in progressing plaques, but they may also be involved in lipoprotein modification during genesis and progression of atherosclerotic lesions.

Enzymatically Modified Nonoxidized Low-Density Lipoprotein Induces Interleukin-8 in Human Endothelial Cells Role of Free Fatty Acids

Background—Treatment of low-density lipoprotein (LDL) with a protease and cholesterolesterase transforms the lipoprotein to an entity that resembles lipoprotein particles in atherosclerotic lesions, which have a high content of free cholesterol, reflecting extensive de-esterification in the intima. Because de-esterification would occur beneath the endothelium, we examined the effects of enzymatically modified LDL (E-LDL) on cultured endothelial cells. Methods and Results—Incubation of endothelial cells with E-LDL provoked selective accumulation of interleukin (IL)-8 mRNA and production of the cytokine. Chemical analyses and depletion experiments indicated that the effect was caused by the presence of free fatty acids in the altered lipoprotein. Reconstitution studies demonstrated that the oleic and linoleic acids associated with E-LDL are particularly effective IL-8 inducers. The effects of E-LDL on endothelial cells could be abrogated with albumin. Conclusion—IL-8 is required for rolling monocytes to adhere firmly to the endothelium; thus, the findings reveal a link between subendothelial entrapment of LDL, cleavage of cholesterol esters, and monocyte recruitment into the lesion.

Enzymatically Modified LDL Induces Cathepsin H in Human Monocytes. Potential Relevance in Early Atherogenesis

Objective—Modification with proteases and cholesterylesterase transforms LDL to a moiety that resembles lipoproteins isolated from atherosclerotic lesions and possesses atherogenic properties. To identify changes in monocyte-derived foam cells laden with enzymatically modified LDL (E-LDL), we compared patterns of the most abundant transcripts in these cells after incubation with LDL or E-LDL. Methods and Results—Serial analyses of gene expression (SAGE) libraries were constructed from human monocytes after treatment with LDL or E-LDL. Several tags were differentially expressed in LDL-treated versus E-LDL–treated cells, whereby marked selective induction by E-LDL of cathepsin H was conspicuous. We show that cathepsin H is expressed in atherosclerotic lesions in colocalization with E-LDL. Furthermore, we demonstrate that LDL modified with cathepsin H and cholesterylesterase can confer onto LDL the capacity to induce macrophage foam cell formation and to induce cathepsin H. Conclusions—Cathepsin H could contribute to the transformation of LDL to an atherogenic moiety; the process might involve a self-sustaining amplifying circle.

One-step polymerase chain reaction-based typing of Helicobacter pylori vacA gene: association with gastric histopathology

Abstract Heterogeneity of the Helicobacter pylori vacA gene may be associated with bacterial virulence and presentation. In this study, the possible correlation between vacA genotypes and gastric histopathology was investigated. Using a modified one-step polymerase chain reaction (PCR)-based method, 122 of 131 H. pylori isolates obtained from 63 of 67 patients from Germany were classified into distinct vacA genotypes according to their signal sequence (s1 or s2) and their midregion alleles (m1 or m2). A possible subtype of m1, now alluded to as m3, was identified in one-third of the isolates. Signal sequence s1 was significantly associated with higher H. pylori density but not with gastric inflammation parameters as compared with s2. Compared with m2, m1 initially appeared to correlate with higher mononuclear cell scores in corpus, although not with H. pylori density. Upon differentiation between m1 and m3, however, only the latter was associated with the high cell scores. Moreover, m3 also correlated with a higher antral H. pylori density. Positive cagA status correlated significantly with vacA signal sequence s1, and higher gastric mononuclear cell scores and corpus neutrophil score. H. pylori density was always associated with enhanced gastric neutrophil and corpus mononuclear cell scores. These data indicate a significant association of specific vacA genotypes with enhanced bacterial density and gastric inflammation. PCR-based identification of the respective alleles can now easily be performed in the diagnostic laboratory using a one-step PCR assay.

Enzymatically hydrolyzed low-density lipoprotein modulates inflammatory responses in endothelial cells.

There is evidence that low-density lipoprotein (LDL) is modified by hydrolytic enzymes, and that the product (E-LDL) induces selective production of interleukin 8 (IL-8) in endothelial cells. Since nuclear factor-kappaB (NF-kappaB) is a major regulator of IL-8 transcription, we studied its activation in endothelial cells treated with E-LDL. Unexpectedly, the modified lipoprotein not only failed to activate NF-kappaB, but completely blocked its activation by tumour necrosis factor-alpha (TNF-alpha) in EA.hy926-cells, as assessed by electrophoretic mobility shift assays and immunofluorescence. Inhibition occurred upstream of NF-kappaB translocation, as inhibitor of NF-kappaB- (IkappaB)-phosphorylation was suppressed by E-LDL. In contrast to NF-kappaB, transcription factor activator protein-1 (AP-1) proved to be activated. Removal of free fatty acids present in E-LDL obliterated both activation of AP-1 and inhibition of NF-kappaB. Chromatin immunoprecipitation revealed that phosphorylated c-jun, but not NF-kappaBp65 bound to the natural IL-8 promoter. Production of endothelial IL-8 and simultaneous modulation of NF-kappaB in response to hydrolyzed LDL might serve to protect the vessel wall and promote silent removal of the insudated lipoprotein.

No association between Helicobacter pylori genotypes and antibiotic resistance phenotypes within families.

Background. Triple therapy combining a proton pump inhibitor with two antibiotics, e.g. clarythromycin (CLR), metronidazole (MTZ) or amoxicillin (AMX), represents the standard in Helicobacter pylori eradication regimens. Resistance to antimicrobial agents, particularly MTZ (up to 56% in Western countries) and CLR (up to 15% in southern Europe), is frequently observed and may be associated with treatment failure [ 1 ]. Recently, several studies indicated that individual H. pylori colonies from a single anatomic site may not always yield identical genotypes, or the identical patterns of susceptibility to antibiotics [ 2–5 ]. Representative for every single patient we analyzed 27 H. pylori antrum isolates for susceptibility to antimicrobial agents in order to test whether identical H. pylori genotypes exhibit a similar pattern of susceptibility to antibiotics.