Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

Emerging approaches for imaging vulnerable plaques in patients

The diagnosis of vulnerable plaques, which have the propensity to develop atherothrombosis, remains an elusive goal in clinical medicine. The most accepted features of vulnerable plaques, such as a large lipid core, increased inflammatory milieu and thin fibrous caps, have been well characterized through pathological studies. The ability to image a vulnerable plaque in susceptible patients would theoretically result in useful prognostic information that can be used to either monitor or treat patients at risk more aggressively. Several invasive techniques, such as integrated backscatter, virtual histology, palpography, optical coherence tomography and thermal heterogeneity, have been validated ex vivo and are now being evaluated in clinical studies. Non-invasive techniques, such as nuclear imaging, show promise in identifying increased metabolic activity and characteristic features of vulnerable plaques in patients. Natural history and intervention studies will need to be performed to determine whether identifying and treating vulnerable plaques will lead to improved clinical outcomes.     

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA⁺ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE⁺ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.     

Structural features of a hyperthermostable endo-beta-1,3-glucanase in solution and adsorbed on "invisible" particles

Conformational characteristics and the adsorption behavior of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133 degrees C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.     

Neutralization of oxidized phospholipids attenuates age‐associated bone loss in mice

Oxidized phospholipids (OxPLs) are pro‐inflammatory molecules that affect bone remodeling under physiological conditions. Transgenic expression of a single‐chain variable fragment (scFv) of the antigen‐binding domain of E06, an IgM natural antibody that recognizes the phosphocholine (PC) moiety of OxPLs, increases trabecular and cortical bone in adult male and female mice by increasing bone formation. OxPLs increase with age, while natural antibodies decrease. Age‐related bone loss is associated with increased oxidative stress and lipid peroxidation and is characterized by a decline in osteoblast number and bone formation, raising the possibility that increased OxPLs, together with the decline of natural antibodies, contribute to age‐related bone loss. We show here that transgenic expression of E06‐scFv attenuated the age‐associated loss of spinal, femoral, and total bone mineral density in both female and male mice aged up to 22 and 24 months, respectively. E06‐scFv attenuated the age‐associated decline in trabecular bone, but not cortical bone, and this effect was associated with an increase in osteoblasts and a decrease in osteoclasts. Furthermore, RNA‐seq analysis showed that E06‐scFv increased Wnt10b expression in vertebral bone in aged mice, indicating that blocking OxPLs increases Wnt signaling. Unlike age‐related bone loss, E06‐scFv did not attenuate the bone loss caused by estrogen deficiency or unloading in adult mice. These results demonstrate that OxPLs contribute to age‐associated bone loss. Neutralization of OxPLs, therefore, is a promising therapeutic target for senile osteoporosis, as well as atherosclerosis and non‐alcoholic steatohepatitis (NASH), two other conditions shown to be attenuated by E06‐scFv in mice.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.     

Impact of Fungicides on the Diversity and Function of Non-target Ammonia-Oxidizing Microorganisms Residing in a Litter Soil Cover

Litter soil cover constitutes an important micro-ecosystem in sustainable viticulture having a key role in nutrient cycling and serving as a habitat of complex microbial communities. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are known to regulate nitrification in soil while little is known regarding their function and diversity in litter. We investigated the effects of two fungicides, penconazole and cyprodinil, commonly used in vineyards, on the function and diversity of total and active AOB and AOA in a microcosm study. Functional changes measured via potential nitrification and structural changes assessed via denaturating gradient gel electrophoresis (DGGE) at the DNA and RNA levels were contrasted with pesticide dissipation in the litter layer. The latter was inversely correlated with potential nitrification, which was temporarily inhibited at the initial sampling dates (0 to 21?days) when nearly 100?% of the applied pesticide amounts was still present in the litter. Fungicides induced changes in AOB and AOA communities with RNA-DGGE analysis showing a higher sensitivity. AOA were more responsive to pesticide application compared to AOB. Potential nitrification was less sensitive to the fungicides and was restored faster than structural changes, which persisted. These results support the theory of microbial redundancy for nitrification in a stressed litter environment.