Lipocalin (LCN) 2 Mediates Pro-Atherosclerotic Processes and Is Elevated in Patients with Coronary Artery Disease

Background

Lipocalin (LCN) 2 is associated with multiple acute and chronic inflammatory diseases but the underlying molecular and cellular mechanisms remain unclear. Here, we investigated whether LCN2 is released from macrophages and contributes to pro-atherosclerotic processes and whether LCN2 plasma levels are associated with the severity of coronary artery disease progression in humans.

Methods and Results

In an autocrine-paracrine loop, tumor necrosis factor (TNF)-α promoted the release of LCN2 from murine bone-marrow derived macrophages (BMDM) and vice versa. Moreover, LCN2 stimulation of BMDM led to up-regulation of M1 macrophage markers. In addition, enhanced migration of monocytic J774A.1 cells towards LCN2 was observed. Furthermore, LCN2 increased the expression of the scavenger receptors Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) as well as scavenger receptor class A-1 (SRA-1) and induced the conversion of macrophages to foam cells. In atherosclerotic lesions of low density lipoprotein receptor-deficient (ldlr ^−/−) mice fed a high fat, high cholesterol diet, LCN2 was found to be co-localized with macrophages in the shoulder region of the atherosclerotic plaque. In addition, LCN2 plasma levels were significantly increased in plasma samples of these mice. Finally, LCN2 plasma levels correlated with the severity of coronary artery disease (CAD) in patients as determined by coronary angiography.

Conclusions

Here we demonstrated that LCN2 plays a pivotal role in processes involved in atherogenesis by promoting polarization and migration of monocytic cells and development of macrophages towards foam cells. Moreover, LCN2 may be used as a prognostic marker to determine the status of CAD progression.     

Improving nematode culture techniques and their effects on amino acid profile with considerations on production costs

The purpose of this study was to investigate the effectiveness of 11 different culture media for production of the free‐living nematode Turbatrix aceti. Several other harvesting methods were tested in addition to mass production. A further focus was the investigation of amino acid alterations caused by the application of various media during the culture of T. aceti and two additional nematode species, Panagrellus redivivus and Caenorhabditis elegans. Finally, a cost analysis for the production of T. aceti was generated and its outcome compared to the production of conventional live feed organisms. Altogether 11 liquid culture media were tested for mass production of the nematode Turbatrix aceti using a minimum of effort in terms of labour and costs. Six harvesting methods, including filtration as well as active swimming of T. aceti were evaluated. Additional to the culture of T. aceti in four of the above‐mentioned media, the nematodes P. redivivus and C. elegans were cultured on two different solid media. Cost analysis for the production of T. aceti includes those of the media, the equipment, as well as the labour costs for culture and harvest. An average density of approx. 30 × 10⁶ ± 8.13 × 10⁶ nematodes L^?1 was achieved for T. aceti. The most efficient method (20 μm filtration) allowed harvesting 85.3 ± 2.7% of the nematodes from the medium without disturbing the particles. Lowest efficiency was achieved by combining sedimentation and filtration, accomplishing a harvest of 42.1 ± 5.8%. The amino acid profile of all three nematode species turned out to be both stable and very similar. Amino acid enrichment had little effect. The costs for producing one million T. aceti individuals ranged between 5.39 and 6.19 €, where labour costs accounted for 73 to 84% of the total production costs. In conclusion, T. aceti appears to be very robust, easy to handle, as well as cheaper to cultivate compared to other live‐feed organisms. Therefore, its use in commercial aquaculture should be given future consideration.     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Gα<Subscript>16</Subscript> interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

Background  

G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα₁₆ as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ).     

A covariance structure model for the admixture of binary genetic variation

I derive a covariance structure model for pairwise linkage disequilibrium (LD) between binary markers in a recently admixed population and use a generalized least-squares method to fit the model to two different data sets. Both linked and unlinked marker pairs are incorporated in the model. Under the model, a pairwise LD matrix is decomposed into two component matrices, one containing LD attributable to admixture, and another containing, in an aggregate form, LD specific to the populations forming the mixture. I use population genetics theory to show that the latter matrix has block-diagonal structure. For the data sets considered here, I show that the number of source populations can be determined by statistical inference on the canonical correlations of the sample LD matrix.     

Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture

Without a scale-down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high-capacity microscale system that is typically used in fed-batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 10⁶ mg/cell/day and 7 × 10 ⁷ cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.