Describing the Cellular Impact of IQOS™ Smoke Extract and Vibration on Human Vocal Fold Fibroblasts.

OBJECTIVES: The isolated or combined effects of vibration and smoke extract (SE) from the IQOS™ "heat-not-burn" technology on human vocal fold fibroblasts (hVFF) were evaluated in an in vitro setting in order to elucidate their influence on vocal fold (patho-) physiology. STUDY DESIGN: Experimental pilot study using intervention with IQOS™-SE in vitro. METHODS: Immortalized hVFF were exposed to IQOS™-SE or control medium under static or vibrational conditions. A phonomimetic bioreactor was used to deliver vibrational patterns to hVFF over a period of 5days. Cytotoxicity was quantified by lactate dehydrogenase assay. Effects on extracellular matrix production, inflammation, fibrogenesis, and angiogenesis were assessed by reverse transcription-quantitative polymerase chain reaction, western blot, enzyme-linked immunosorbent assay, and Magnetic Luminex assays. RESULTS: We observed significant changes induced either by IQOS™-SE exposure alone (matrix metalloproteinase 1, fibronectin, cyclooxygenase (COX)1, interleukin-8 gene expression), or by the combination of IQOS™-SE and vibration (hyaluronidase 2, COX2, interleukin-8 protein levels, vascular endothelial growth factor D). CONCLUSION: Short-term in vitro exposure of hVFF to IQOS™-SE did not result in cytotoxicity and reduced the gene expression of measured inflammation mediators, but had no effect on their protein expression. However, the clinical effects of long-term IQOS™ use are still not known and further research is needed in order to assess, if IQOS™ is in fact less harmful than conventional cigarettes.

Vocal fold fibroblasts and exposure to vibration in vitro: Does sex matter?

Studies have shown that certain vocal fold pathologies are more common in one sex than the other. This is often explained by differences in the composition of the lamina propria and anatomical differences between female and male vocal folds, resulting in e.g. different fundamental frequencies. Here, we investigated a potential sex-specific voice frequency effect in an in vitro setting using vocal fold fibroblasts from one male and one female donor with and without cigarette smoke extract (CSE) addition. After exposure to either male or female vibration frequency with or without CSE, cells and supernatants were harvested. Gene and protein analysis were performed by means of qPCR, western blot, ELISA and Luminex. We found that exposure of cells to both male and female vibration pattern did not elicit significant changes in the expression of extracellular matrix-, inflammation-, and fibrosis-related genes, compared to control cells. The addition of CSE to vibration downregulated the gene expression of COL1A1 in cells exposed to the female vibration pattern, as well as induced MMP1 and PTGS2 in cells exposed to both female and male vibration pattern. The protein expression of MMP1 and COX2 was found to be significantly upregulated only in cells exposed to CSE and female vibration pattern. To conclude, different vibration patterns alone did not cause different responses of the cells. However, the female vibration pattern in combination with CSE had a tendency to elicit/maintain more pro-inflammatory responses in cells than the male vibration pattern.

Bilateral Functional Electrical Stimulation for the Treatment of Presbyphonia in a Sheep Model.

OBJECTIVES: The aim of the study was to increase muscle volume and improve phonation characteristics of the aged ovine larynx by functional electrical stimulation (FES) using a minimally invasive surgical procedure. METHODS: Stimulation electrodes were placed bilaterally near the terminal adduction branch of the recurrent laryngeal nerves (RLN). The electrodes were connected to battery powered pulse generators implanted subcutaneously at the neck region. Training patterns were programmed by an external programmer using a bidirectional radio frequency link. Training sessions were repeated automatically by the implant every other day for 1 week followed by every day for 8 weeks in the awake animal. Another group of animals were used as sham, with electrodes positioned but not connected to an implant. Outcome parameters included gene expression analysis, histological assessment of muscle fiber size, functional analysis, and volumetric measurements based on three-dimensional reconstructions of the entire thyroarytenoid muscle (TAM). RESULTS: Increase in minimal muscle fiber diameter and an improvement in vocal efficiency were observed following FES, compared with sham animals. CONCLUSION: This is the first study to demonstrate beneficial effects in the TAM of FES at molecular, histological, and functional levels. FES of the terminal branches of the RLN reversed the effects of age-related changes and improved vocal efficiency. LEVEL OF EVIDENCE: NA Laryngoscope, 134:848-854, 2024.

Exploring the anti-inflammatory potential of topical hyaluronic acid for vocal fold injury in a rat model.

PURPOSE: Vocal fold injuries are associated with fibrosis and dysphonia, which is a major obstacle to surgical treatment. The aim of this study is to evaluate the effect of topical hyaluronic acid with or without diclofenac on the inflammatory phase of vocal fold wound healing. METHODS: Forty-one male Sprague-Dawley rats were randomly assigned to four groups: an uninjured control group, an injured control group without any treatment, and two intervention groups in which hyaluronic acid with or without diclofenac was applied to the injured vocal fold. Gene expression of inflammatory markers and ECM-related molecules were examined. RESULTS: Vocal fold injury resulted in a significant upregulation of inflammatory parameters [Ptgs2, Il1b and Il10] and Has1. Tgfb1, Has3 and Eln gene expression were significantly downregulated by the topical application of hyaluronic acid. The combination of hyaluronic acid and diclofenac did not result in any significant changes. CONCLUSIONS: Vocal fold wound healing was significantly improved by a single post-operative topical application of hyaluronic acid. The addition of diclofenac may provide no additional benefit.

Comparing Effects of Short- and Long-Term Exposure of Cigarette Smoke Extract on Human Vocal Fold Fibroblasts.

OBJECTIVES: To explore the effects of short- and long-term cigarette smoke extract (CSE) stimulation on the expression of extracellular matrix (ECM) components and inflammatory cytokines in an in vitro model for studying Reinke's edema using human vocal fold fibroblasts (hVFF). STUDY DESIGN: Experimental pilot study using intervention with CSE in vitro. METHODS: Immortalized hVFF were pretreated with 5% CSE or control medium over a period of 2 or 8 weeks, followed by a final 3-day incubation time. We evaluated cell proliferation and examined gene and protein expression of control- and CSE-treated cells using quantitative polymerase chain reaction, Western Blot and enzyme linked immunosorbent assay. RESULTS: Cell numbers of CSE-treated hVFF strongly decreased after 8 weeks and limited the overall duration of the experiment. We observed significant upregulations in gene expression and protein levels of inflammatory markers (cyclooxygenase COX1, COX2) and ECM components (decorin, matrix metalloproteinase 1, transglutaminase 2, gremlin 2) induced by CSE after 2 and 8 weeks. Interleukin 1 receptor 1, prostaglandin I2 synthase, collagen- and hyaluronan-related gene expression showed minor upregulations. The majority of the observed genes were similarly regulated at both time points. However, the CSE-induced mRNA level of COX1 was ablated after 8 weeks. CONCLUSION: Long-term treatment did not yield results significantly different from the short-term protocol. Therefore, we propose that prolonged CSE exposure is not superior to short-term settings, which save both time and materials.

Introducing a new type of alternative laryngeal mucosa model.

Research of human vocal fold (VF) biology is hampered by several factors. The sensitive microstructure of the VF mucosa is one of them and limits the in vivo research, as biopsies carry a very high risk of scarring. A laryngeal organotypic model consisting of VF epithelial cells and VF fibroblasts (VFF) may overcome some of these limitations. In contrast to human VFF, which are available in several forms, availability of VF epithelial cells is scarce. Buccal mucosa might be a good alternative source for epithelial cells, as it is easily accessible, and biopsies heal without scarring. For this project, we thus generated alternative constructs consisting of immortalized human VF fibroblasts and primary human buccal epithelial cells. The constructs (n = 3) were compared to native laryngeal mucosa at the histological and proteomic level. The engineered constructs reassembled into a mucosa-like structure after a cultivation period of 35 days. Immunohistochemical staining confirmed a multi-layered stratified epithelium, a collagen type IV positive barrier-like structure resembling the basement membrane, and an underlying layer containing VFF. Proteomic analysis resulted in a total number of 1961 identified and quantified proteins. Of these, 83.8% were detected in both native VF and constructs, with only 53 proteins having significantly different abundance. 15.3% of detected proteins were identified in native VF mucosa only, most likely due to endothelial, immune and muscle cells within the VF samples, while 0.9% were found only in the constructs. Based on easily available cell sources, we demonstrate that our laryngeal mucosa model shares many characteristics with native VF mucosa. It provides an alternative and reproducible in vitro model and offers many research opportunities ranging from the study of VF biology to the testing of interventions (e.g. drug testing).

Effect of functional electric stimulation on phonation in an ex vivo aged ovine model.

With age, the atrophy of the thyroarytenoid muscle (TAM), and thus atrophy of the vocal folds, leads to decreased glottal closure, increased breathiness, and a loss in voice quality, which results in a reduced quality of life. A method to counteract the atrophy of the TAM is to induce hypertrophy in the muscle by functional electric stimulation (FES). In this study, phonation experiments were performed with ex vivo larynges of six stimulated and six unstimulated ten-year-old sheep to investigate the impact of FES on phonation. Electrodes were implanted bilaterally near the cricothyroid joint. FES treatment was provided for nine weeks before harvesting. The multimodal measurement setup simultaneously recorded high-speed video of the vocal fold oscillation, the supraglottal acoustic signal, and the subglottal pressure signal. Results of 683 measurements show a 65.6% lower glottal gap index, a 22.7% higher tissue flexibility (measured by the amplitude to length ratio), and a 473.7% higher coefficient of determination (R2) of the regression of subglottal and supraglottal cepstral peak prominence during phonation for the stimulated group. These results suggest that FES improves the phonatory process for aged larynges or presbyphonia.

Descriptive proteomics of paired human vocal fold and buccal mucosa tissue.

The vast majority of voice disorders is associated with changes of the unique, but delicate, human vocal fold mucosa. The ability to develop new effective treatment methods is significantly limited by the physical inaccessibility and the extremely rare occasions under which healthy tissue biopsies can be obtained. Therefore, the interest in laryngological research has shifted to human oral (buccal) mucosa, a similar and more easily available tissue. The harvesting process is less invasive and accompanied with faster healing and less scarring, compared to vocal fold mucosa. Here we report a descriptive proteomic comparison of paired human buccal and vocal fold mucosa by high-resolution mass spectrometry (CID-MS/MS). Our study identified a total of 1575 proteins detected within both tissues that are highly consistent in several crucial biological processes, cellular components, and molecular functions. Hence, our proteomic analysis will provide a fundamental resource for the laryngological research community.

Vocal Fold Fibroblasts in Reinke's Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control.

The voice disorder Reinke's edema (RE) is a smoking- and voice-abuse associated benign lesion of the vocal folds, defined by an edema of the Reinke's space, accompanied by pathological microvasculature changes and immune cell infiltration. Vocal fold fibroblasts (VFF) are the main cell type of the lamina propria and play a key role in the disease progression. Current therapy is restricted to symptomatic treatment. Hence, there is an urgent need for a better understanding of the molecular causes of the disease. In the present study, we investigated differential expression profiles of RE and control VFF by means of RNA sequencing. In addition, fast gene set enrichment analysis (FGSEA) was performed in order to obtain involved biological processes, mRNA and protein levels of targets of interest were further evaluated. We identified 74 differentially regulated genes in total, 19 of which were upregulated and 55 downregulated. Differential expression analysis and FGSEA revealed upregulated genes and pathways involved in extracellular matrix (ECM) remodeling, inflammation and fibrosis. Downregulated genes and pathways were involved in ECM degradation, cell cycle control and proliferation. The current study addressed for the first time a direct comparison of VFF from RE to control and evaluated immediate functional consequences.

Agonism of Prostaglandin E2 Receptor 4 Ameliorates Tubulointerstitial Injury in Nephrotoxic Serum Nephritis in Mice.

Selectively targeting the E-type prostanoid receptor 4 (EP4) might be a new therapeutic option in the treatment of glomerulonephritis (GN), since the EP4 receptor is expressed on different immune cells, resident kidney cells, and endothelial cells, which are all involved in the pathogenesis of immune-complex GN. This study aimed to evaluate the therapeutic potential and to understand the mode of action of EP4 agonist in immune-complex GN using the murine model of nephrotoxic serum nephritis (NTS). In vivo, NTS mice were treated two times daily with two different doses of an EP4 agonist ONO AE1-329 or vehicle for 14 days total. The effect of PGE2 and EP4 agonism and antagonism was tested on murine distal convoluted tubular epithelial cells (DCT) in vitro. In vivo, the higher dose of the EP4 agonist led to an improved NTS phenotype, including a reduced tubular injury score and reduced neutrophil gelatinase-associated lipocalin (NGAL) and blood urea nitrogen (BUN) levels. EP4 agonist treatment caused decreased CD4+ T cell infiltration into the kidney and increased proliferative capacity of tubular cells. Injection of the EP4 agonist resulted in dose-dependent vasodilation and hypotensive episodes. The low-dose EP4 agonist treatment resulted in less pronounced episodes of hypotension. In vitro, EP4 agonism resulted in cAMP production and increased distal convoluted tubular (DCT) proliferation. Taken together, EP4 agonism improved the NTS phenotype by various mechanisms, including reduced blood pressure, decreased CD4+ T cell infiltration, and a direct effect on tubular cells leading to increased proliferation probably by increasing cAMP levels.

Exploring the Pathophysiology of Reinke's Edema: The Cellular Impact of Cigarette Smoke and Vibration.

OBJECTIVES: To explore the isolated or combined effects of cigarette smoke extract (CSE) and vibration on human vocal fold fibroblasts (hVFF) in an in vitro setting in order to elucidate their influence in the pathophysiology of Reinke's edema (RE). STUDY DESIGN: Immortalized hVFF were exposed to CSE or control medium under static or vibrational conditions. A phonomimetic bioreactor was used to deliver vibrational patterns to hVFF over a period of 5 days. METHODS: Cytotoxicity was quantified using a lactate dehydrogenase assay. We employed reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Magnetic Luminex(R) assays (R&D Systems, Minneapolis, MN) to assess the influence on extracellular matrix production, fibrogenesis, inflammation, and angiogenesis. RESULTS: We observed significant changes induced by CSE alone (hyaluronic acid, matrix metalloproteinase 1, Interleukin-8, cyclooxygenase [COX]1, COX2, vascular endothelial growth factor [VEGF]D), as well as settings in which only the combination of CSE and vibration led to significant changes (transforming growth factor beta 1, VEGFA, VEGFC). Also, CSE-induced levels of COX2 were only significantly reduced when vibration was applied. CONCLUSION: We were able to explore the cellular effects of CSE and vibration on hVFF by employing a phonomimetic bioreactor. Whereas cigarette smoke is generally accepted as a risk factor for RE, the role of vibration remained unclear as it is difficult to study in humans. Our data showed that some genes and proteins in the pathophysiological context of RE were only affected when CSE in combination with vibration was applied. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E547-E554, 2021.

In vitro mechanical vibration down-regulates pro-inflammatory and pro-fibrotic signaling in human vocal fold fibroblasts.

INTRODUCTION: Voice rest following phonotrauma or phonosurgery has a considerable clinical impact, but clinical recommendations are inconsistent due to inconclusive data. As biopsies of the vocal folds (VF) for molecular biology studies in humans are unethical, we established a new in vitro model to explore the effects of vibration on human vocal fold fibroblasts (hVFF) in an inflammatory and normal state, which is based on previously published models. METHODS: By using a phonomimetic bioreactor we were able to apply predefined vibrational stress patterns on hVFF cultured under inflammatory or normal conditions. Inflammatory and pro-fibrotic stimuli were induced by interleukin (IL)1ß and transforming growth factor (TGF)ß1, respectively. Mechanical stimulation was applied four hours daily, over a period of 72 hours. Outcome measurements comprised assessment of extracellular matrix (ECM)-related components, angiogenic factors, and inflammatory and fibrogenic markers on gene expression and protein levels. RESULTS: Under inflammatory conditions, the inflammatory cytokine IL11, as well as the myofibroblast marker alpha smooth muscle actin (α-SMA) were significantly reduced when additional vibration was applied. The desirable anti-fibrotic ECM component hyaluronic acid was increased following cytokine treatment, but was not diminished following vibration. CONCLUSION: Our experiments revealed the effect of vibrational stress on hVFF in an inflammatory state. Elevated levels of certain pro-inflammatory/pro-fibrotic factors could be mitigated by additional vibrational excitation in an in vitro setting. These findings corroborate clinical studies which recommend early voice activation following an acute event.

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema.

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.

Development and validation of a novel phonomimetic bioreactor.

Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold's lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFß-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device.

MicroRNA-142-3p improves vascular relaxation in uremia.

BACKGROUND AND AIMS: Chronic kidney disease (CKD) is strongly associated with a high burden of cardiovascular morbidity and mortality. Therefore, we aimed to characterize the putative role of microRNAs (miR)s in uremic vascular remodelling and endothelial dysfunction. METHODS: We investigated the expression pattern of miRs in two independent end-stage renal disease (ESRD) cohorts and in the animal model of uremic DBA/2 mice via quantitative RT-PCR. Moreover, DBA/2 mice were treated with intravenous injections of synthetic miR-142-3p mimic and were analysed for functional and morphological vascular changes by mass spectrometry and wire myography. RESULTS: The expression pattern of miRs was regulated in ESRD patients and was reversible after kidney transplantation. Out of tested miRs, only blood miR-142-3p was negatively associated with carotid-femoral pulse-wave velocity in CKD 5D patients. We validated these findings in a murine uremic model and found similar suppression of miR-142-3p as well as decreased acetylcholine-mediated vascular relaxation of the aorta. Therefore, we designed experiments to restore bioavailability of aortic miR-142-3p in vivo via intravenous injection of synthetic miR-142-3p mimic. This intervention restored acetylcholine-mediated vascular relaxation. CONCLUSIONS: Taken together, we provide compelling evidence, both in humans and in mice, that miR-142-3p constitutes a potential pharmacological agent to prevent endothelial dysfunction and increased arterial stiffness in ESRD.

Blockade of prostaglandin E2 receptor 4 ameliorates nephrotoxic serum nephritis.

Prostaglandin E2 (PGE2) signaling is known to modulate inflammation and vascular resistance. Receptors of PGE2 [E-type prostanoid receptors (EP)] might be an attractive pharmacological target in immune-mediated diseases such as glomerulonephritis. We hypothesized that selective EP4 antagonism improves nephrotoxic serum nephritis (NTS) by its anti-inflammatory properties. Mice were subjected to NTS and treated with the EP4 antagonist ONO AE3-208 (10 mg·kg body wt-1·day-1] or vehicle starting from disease initiation. In one set of experiments, treatment was started 4 days after NTS induction. Tubular epithelial cells were evaluated in vitro under starving conditions. EP4 antagonist treatment significantly improved the NTS phenotype without affecting blood pressure levels. Remarkably, the improved NTS phenotype was also observed when treatment was started 4 days after NTS induction. EP4 antagonism decreased tubular chemokine (C-X-C motif) ligand ( Cxcl) 1 and Cxcl-5 expression and thereby subsequently reduced interstitial neutrophil infiltration into the kidney. In vitro, tubular epithelial cells increasingly expressed Cxcl-5 mRNA and Cxcl-5 protein when treated with PGE2 or an EP4 agonist under starving conditions, which was blunted by EP4 antagonist treatment. Together, EP4 antagonism improves the NTS phenotype, probably by decreasing mainly Cxcl-5 production in tubular cells, thereby reducing renal neutrophil infiltration.

RNA editing of Filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure.

Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.

Gestational diabetes mellitus modulates cholesterol homeostasis in human fetoplacental endothelium.

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H2DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography-mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [3H]-cholesterol labeled HPEC and [14C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7ß-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL3 (78 ±â€¯17%, 40 ±â€¯9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.

Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS).

Mitochondrial Ca2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO•) production. However, it is not entirely clear if the organelles support or counteract NO• biosynthesis by taking up Ca2+. The objective of this study was to verify whether or not mitochondrial Ca2+ uptake influences Ca2+-triggered NO• generation by endothelial NO• synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO• probes, the geNOps, and Ca2+ sensors to monitor single cell NO• and Ca2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP3)-generating agonist. Mitochondrial Ca2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca2+-triggered NO• increase independently of global cytosolic Ca2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca2+ sequestration and Ca2+-induced NO• signals. The positive correlation between mitochondrial Ca2+ elevation and NO• production was independent of eNOS phosphorylation at serine1177. Our findings emphasize that manipulating mitochondrial Ca2+ uptake may represent a novel strategy to control eNOS-mediated NO• production.

Heterogeneous susceptibility for uraemic media calcification and concomitant inflammation within the arterial tree.

BACKGROUND: End-stage renal disease (ESRD) is strongly associated with arterial calcification of the tunica media, decreased vascular compliance and sudden cardiac death. Here, we analysed the distribution pattern of uraemic media calcification and concomitant inflammation in mice and men. METHODS: Uraemia was induced in DBA/2 mice with high-phosphate diet. Subsequently, we analysed arterial medial calcification using histology, mass spectrometry, and wire myography. Gene expression was quantified using a whole transcriptome array and quantitative PCR. In a cohort of 36 consecutive patients with CKD stage 4-5, we measured the calcium score of the coronary arteries, the ascending thoracic aorta and the infrarenal abdominal aorta using computed tomography scans. RESULTS: Uraemic DBA/2 mice showed only minor calcifications in thoracic aortas, whereas there was overt media calcification in abdominal aortas. The transcriptional profile and immunohistochemistry revealed induction of Vcam1 expression by vascular smooth muscle cells in uraemic abdominal aortas. Macrophages infiltrated the tunica media of the abdominal aorta. Anti-inflammatory treatment did not improve uraemic media calcification in our animal model. Arterial calcifications in ESRD patients showed a similar distribution pattern in computed tomography scans, with higher calcium scores of the abdominal aorta when compared with the thoracic aorta. CONCLUSION: Taken together, there was a similar heterogeneous pattern of calcification in both mice and humans, where the abdominal aorta was more prone to media calcification when compared with the thoracic aorta. In uraemia, smooth muscle cells of the abdominal aorta showed a phenotypic switch to an inflammatory and osteoblastic phenotype.