Lipid loading of human vascular smooth muscle cells induces changes in tropoelastin protein levels and physical structure

Aggregated low-density lipoprotein (agLDL), one of the main LDL modifications in the arterial intima, contributes to massive intracellular cholesteryl ester (CE) accumulation in human vascular smooth muscle cells (VSMC), which are major producers of elastin in the vascular wall. Our aim was to analyze the levels, physical structure, and molecular mobility of tropoelastin produced by agLDL-loaded human VSMC (agLDL-VSMC) versus that produced by control VSMC. Western blot analysis demonstrated that agLDL reduced VSMC-tropoelastin protein levels by increasing its degradation rate. Moreover, our results demonstrated increased levels of precursor and mature forms of cathepsin S in agLDL-VSMC. Fourier transform infrared analysis revealed modifications in the secondary structures of tropoelastin produced by lipid-loaded VSMCs. Thermal and dielectric analyses showed that agLDL-VSMC tropoelastin has decreased glass transition temperatures and distinct chain dynamics that, in addition to a loss of thermal stability, lead to strong changes in its mechanical properties. In conclusion, agLDL lipid loading of human vascular cells leads to an increase in cathepsin S production concomitantly with a decrease in cellular tropoelastin protein levels and dramatic changes in secreted tropoelastin physical structure. Therefore, VSMC-lipid loading likely determines alterations in the mechanical properties of the vascular wall and plays a crucial role in elastin loss during atherosclerosis.     

Relationship between Audiometric slope and tinnitus pitch in tinnitus patients: insights into the mechanisms of tinnitus generation

Background

Different mechanisms have been proposed to be involved in tinnitus generation, among them reduced lateral inhibition and homeostatic plasticity. On a perceptual level these different mechanisms should be reflected by the relationship between the individual audiometric slope and the perceived tinnitus pitch. Whereas some studies found the tinnitus pitch corresponding to the maximum hearing loss, others stressed the relevance of the edge frequency. This study investigates the relationship between tinnitus pitch and audiometric slope in a large sample.

Methodology

This retrospective observational study analyzed 286 patients. The matched tinnitus pitch was compared to the frequency of maximum hearing loss and the edge of the audiogram (steepest hearing loss) by t-tests and correlation coefficients. These analyses were performed for the whole group and for sub-groups (uni- vs. bilateral (117 vs. 338 ears), pure-tone vs. narrow-band (340 vs. 115 ears), and low and high audiometric slope (114 vs. 113 ears)).

Findings

For the right ear, tinnitus pitch was in the same range and correlated significantly with the frequency of maximum hearing loss, but differed from and did not correlate with the edge frequency. For the left ear, similar results were found but the correlation between tinnitus pitch and maximum hearing loss did not reach significance. Sub-group analyses (bi- and unilateral, tinnitus character, slope steepness) revealed identical results except for the sub-group with high audiometric slope which revealed a higher frequency of maximum hearing loss as compared to the tinnitus pitch.

Conclusion

The study-results confirm a relationship between tinnitus pitch and maximum hearing loss but not to the edge frequency, suggesting that tinnitus is rather a fill-in-phenomenon resulting from homeostatic mechanisms, than the result of deficient lateral inhibition. Sub-group analyses suggest that audiometric steepness and the side of affected ear affect this relationship. Future studies should control for these potential confounding factors.     

Metabolomics of dietary Fatty Acid restriction in patients with phenylketonuria

Background

Patients with phenylketonuria (PKU) have to follow a lifelong phenylalanine restricted diet. This type of diet markedly reduces the intake of saturated and unsaturated fatty acids especially long chain polyunsaturated fatty acids (LC-PUFA). Long-chain saturated fatty acids are substrates of mitochondrial fatty acid oxidation for acetyl-CoA production. LC-PUFA are discussed to affect inflammatory and haemostaseological processes in health and disease. The influence of the long term PKU diet on fatty acid metabolism with a special focus on platelet eicosanoid metabolism has been investigated in the study presented here.

Methodology/Principal Findings

12 children with PKU under good metabolic control and 8 healthy controls were included. Activated fatty acids (acylcarnitines C6–C18) in dried blood and the cholesterol metabolism in serum were analyzed by liquid chromatographic tandem mass spectrometry (LC-MS/MS). Fatty acid composition of plasma glycerophospholipids was determined by gas chromatography. LC-PUFA metabolites were analyzed in supernatants by LC-MS/MS before and after platelet activation and aggregation using a standardized protocol. Patients with PKU had significantly lower free carnitine and lower activated fatty acids in dried blood compared to controls. Phytosterols as marker of cholesterol (re-) absorption were not influenced by the dietary fatty acid restriction. Fatty acid composition in glycerophospholipids was comparable to that of healthy controls. However, patients with PKU showed significantly increased concentrations of y-linolenic acid (C18:3n-6) a precursor of arachidonic acid. In the PKU patients significantly higher platelet counts were observed. After activation with collagen platelet aggregation and thromboxane B₂ and thromboxane B₃ release did not differ from that of healthy controls.

Conclusion/Significance

Long-term dietary fatty acid restriction influenced the intermediates of mitochondrial beta-oxidation. No functional influence on unsaturated fatty acid metabolism and platelet aggregation in patients with PKU was detected.     

Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H+-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H+, CHR1 also conducts Na+, K+, and Ca2+. The kinetic selectivity analysis demonstrates that H+ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1505 and CHR1463, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.     

High-impact sulfur compounds: constitutional and configurational assignment of sulfur-containing heterocycles

To unambiguously identify their structures and to evaluate their organoleptic properties, several constitutional und configurational isomers of dialkyl-tetrathianes and dialkyl-pentathiepanes were synthesized by two different synthetic protocols, and separated by preparative gas chromatography. Raman and NMR spectroscopy were used to differentiate between the constitutional 3,6-dialkyl-1,2,4,5-tetrathiane and the 4,6-dialkyl-1,2,3,5-tetrathiane isomers. Furthermore, cis- and trans-isomers of 3,6-dialkyl-1,2,4,5-tetrathianes were distinguished by temperature-dependent NMR experiments. Static, quantum-chemical simulations of the NMR spectra for these cis- and trans-isomers were calculated in the gas layer in order to confirm our experimental assignments. In addition, the assignment of 4,7-alkyl-1,2,3,5,6-pentathiepanes were deducted from their Raman spectra. Dialkyl-tetrathianes and dialkyl-pentathiepanes are interesting components to be used in flavor applications due to their unique olfactory impact and facets.     

Urinary protein profiling with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry in ETB receptor-deficient rats

The pathways leading to salt-sensitive hypertension and renal damage in rescued ETB receptor-deficient (ETBRd) rats are still unknown. The objective of the study was therefore to identify modifications of urinary peptide and protein expression in ETBRd rats (n = 9) and wild-type controls (n = 6) using SDS - polyacrylamide gel electrophoresis (SDS-PAGE) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Glomerular filtration rate, glomerulosclerosis, and tubulointerstitial fibrosis did not differ between the groups. ETBRd rats showed slightly higher blood pressure (p < 0.001), media/lumen ratio of intrarenal arteries (p < 0.01), and albuminuria (p < 0.01). SDS-PAGE confirmed albuminuria, but showed no differences in the urinary excretion of low molecular weight proteins (<60 kDa). SELDI-TOF-MS profiling revealed 9 proteomic features at molecular masses (Da) of 2720, 2980, 3130, 3345, 6466, 6682, 8550, 18 729, and 37 492, which were significantly elevated (p < 0.02) in urine of ETBRd rats. The results demonstrate that, independent of structural changes in the kidneys, ETB-receptor deficiency causes specific differences in urinary peptide and protein excretion. SELDI-TOF-MS may be a valuable tool for the characterization of urinary biomarkers helping to uncover the mechanism of ETBR action in the kidney.     

Inhalation of an endothelin receptor A antagonist attenuates pulmonary inflammation in experimental acute lung injury

We recently demonstrated that inhalation of the endothelin receptor A (ETA) antagonist LU 135252 improved arterial oxygenation and reduced pulmonary artery pressure in experimental acute lung injury (ALI). In this study we analyzed potential immune modulatory effects of inhaled LU 135252 in experimental ALI. ALI was induced by repeated lung lavage in intubated (100% O2) and anesthetized piglets. Animals were randomly assigned to inhale either nebulized LU 135252 (0.3 mg.kg-1, ALI + LU group, n = 8) or saline buffer (ALI control group, n = 16), both for 30 min. Surviving animals were sacrificed 6 h after induction of ALI, and lung tissue specimens were obtained from all animals for histology and immunhistochemistry. Induction of ALI significantly decreased arterial oxygenation in all animals. Inhalation of LU 135252 significantly reduced mortality and induced significant and sustained increase in Pao2 (316 +/- 47 mm Hg vs. control 53 +/- 3 mm Hg, p < 0.001). We measured a significant reduction in the number of pulmonary leukocyte L1 antigen-positive cells in ALI + LU animals (8% +/- 1% positive cells vs. control 12% +/- 2% positive cells, p < 0.05). The number of CD3-positive cells was not altered by treatment with LU 135252. Pulmonary tissue concentration of IL-6 was significantly suppressed by LU 135252 inhalation (4 +/- 1 pg.100 mg-1 wet weight vs. control 7 +/- 1 pg.100 mg-1 wet weight, p < 0.05). Concentrations of TNF-alpha, IL-1beta, and ET-1 in pulmonary tissue were not influenced by inhalation of LU 135252. In conclusion, we demonstrated that inhalation of LU 135252 not only improves mortality and gas exchange, but also blunts the local immune response in experimental ALI.     

Pulmonary fibrosis in L-NAME-treated mice is dependent on an activated endothelin system

Activation of the endothelin (ET) system promotes vasoconstriction, inflammation, and fibrosis in various tissues, including the lung. Therefore, ET-1 transgenic mice overexpressing ET-1 develop pulmonary fibrosis in a slow, age-dependent manner. In vivo, NO is the most important counterregulatory mediator of the ET system and decreases ET-1 promoter activity. The aim of our study was to elucidate the impact on pulmonary inflammation and fibrosis of the interaction between NO and the ET system in young ET-1 transgenic mice before the onset of pulmonary fibrosis. Male ET-1 transgenic mice and wild-type littermates at the age of 8 weeks were randomly allocated to the following 6 groups: WT (n = 11), wild-type animals without treatment; WT + l-NAME (n = 14), wild-type animals receiving l-NAME, an inhibitor of NO synthase; WT + l-NAME + LU (n = 13), wild-type animals receiving l-NAME and LU 302872, a dual ETA/ETB-receptor antagonist; ET1tg (n = 10), ET-1 transgenic mice; ET1tg + l-NAME (n = 13); and ET1tg + l-NAME + LU (n = 13). After 6 weeks, animals were euthanized, and hearts and lungs were harvested for histology and immunohistochemistry. No differences in pulmonary inflammation, as indicated by macrophage infiltration, or in interstitial fibrosis were observed between WT and ET1tg mice at baseline; however, inflammation and interstitial fibrosis were significantly enhanced in ET1tg mice, but not in WT groups, after l-NAME treatment. The combined ETA/ETB-receptor antagonist LU 302872 abolished inflammation and interstitial fibrosis in l-NAME-treated ET1tg mice. Perivascular fibrosis and media/lumen ratio of pulmonary bronchi and arteries did not differ between all study groups. In our study l-NAME induced pulmonary fibrosis and inflammation only in young ET1tg mice. Additional treatment with LU 302872 abolished these effects. We thus conclude that an imbalance between an activated ET system and a suppressed NO system contributes to pulmonary inflammation and fibrosis.