Metabolic plasticity of vascular smooth muscle cells in vascular disease

Unlike cardiac or skeletal muscle cells, vascular smooth muscle cells (VSMCs) retain a remarkable degree of plasticity. On environmental cues they can dedifferentiate from a quiescent contractile state towards phenotypes of increased proliferation and migration as well as secretory capacity and inflammation. This ability to transition between different phenotypes is a prerequisite for physiological vascular remodeling processes, but also plays a key role in the pathogenesis of virtually all vascular diseases. These vascular diseases, above all atherosclerosis resulting in myocardial infarction or stroke, are still the leading cause of death worldwide. Based on the respective metabolic requirements of proliferating versus quiescent cells it was hypothesised that VSMCs undergo metabolic changes during dedifferentiation. Therefore, the aim of this study was to investigate the metabolic adaptions VSMCs exhibit during phenotypic transition and identify possible regulators thereof. Utilising two in vitro models and one in vivo model for VSMC dedifferentiation, this study showed that dedifferentiated VSMCs shift their energy generation from mitochondrial respiration towards elevated glycolysis and lactate production, reminiscent of the Warburg effect observed in cancer cells. Dedifferentiated VSMCs also displayed reduced expression of genes involved in mitochondrial respiration, lower mitochondrial abundance and altered mitochondrial shape, indicating a strong association between mitochondrial homeostasis and VSMC plasticity. The second objective of this study was to investigate whether intervention in VSMC metabolism would affect VSMC plasticity and vascular remodeling. Two known regulators of metabolism, Sirt6 and Sirt7, were chosen for their regulatory function in glucose and mitochondrial metabolism, respectively. The effects of VSMC specific knock-outs of Sirt6 and Sirt7 on VSMC dedifferentiation and proliferation were assessed in vitro and in vivo. Both sirtuins were expected to display atheroprotective functions. This could be confirmed for Sirt7 as the VSMC-specific knock-out of Sirt7 resulted in elevated neointima formation in a carotid artery ligation mouse model and increased plaque sizes in an ApoE-/- atherosclerosis mouse model. VSMC-specific knock-out of Sirt6 did not impact both these parameters. Contrary to expectations, the effects of the Sirt7 knock-out did not seem to be mediated by regulation of mitochondrial homeostasis. The atheroprotective role shown in this study, nevertheless renders Sirt7 an interesting target for the treatment of vascular diseases

Creating tissue with intervertebral disc-like characteristics using gdf5 functionalized silk scaffolds and human mesenchymal stromal cells

For years, researchers have searched for a suitable biomaterial to regenerate the intervertebral disc (IVD). A promising candidate is silk, as there have been several approaches in the past where silk fibroin was used to repair the IVD’s nucleus pulposus (NP) and annulus fibrosus (AF). However, to date, nobody has attempted to recreate IVD tissue with dimensions and cell densities comparable to a human IVD using silk and human mesenchymal stromal cells (MSC). Therefore, silk scaffolds were produced from Bombyx mori yarn. To mimic the AF, the yarn was embroidered into a ring-like structure or patch. To mimic the NP, fibre-additive manufacturing was applied to create highly porous constructs. Half of the NP scaffolds were functionalized with the growth differentiation factor 5 (GDF5). The scaffolds were seeded with MSCs from five human donors in a density of one-third of the density found in the human IVD and cultured for 7, 14 or 21 days in transforming growth factor β1 (TGF-β1)-enriched medium. All scaffolds were biocompatible as cell numbers increased by a factor 4-5. Furthermore, the scaffolds generally showed an anabolic phenotype, which was positively influenced by GDF5, and tissue-like characteristics were promoted based on the scaffolds’ morphology. In conclusion, the here proposed silk scaffolds showed IVD-like characteristics with a size and cell density comparable to human IVD tissue

The use of moderate hypothermia during cardiac surgery is associated with repression of tumour necrosis factor-α via inhibition of activating protein-1: an experimental study

INTRODUCTION: The use of moderate hypothermia during experimental cardiac surgery is associated with decreased expression of tumour necrosis factor (TNF)-α in myocardium and with myocardial protection. In order to identify the cellular mechanisms that lead to that repression, we investigated the effect of hypothermia during cardiac surgery on both main signalling pathways involved in systemic inflammation, namely the nuclear factor-κB (NF-κB) and activating protein-1 pathways. METHOD: Twelve female pigs were randomly subjected to standardized cardiopulmonary bypass with moderate hypothermia or normothermia (temperature 28°C and 37°C, respectively; six pigs in each group). Myocardial probes were sampled from the right ventricle before, during and 6 hours after bypass. We detected mRNA encoding TNF-α by competitive RT-PCR and measured protein levels of TNF-α, inducible nitric oxide synthase and cyclo-oxygenase-2 by Western blotting. Finally, we assessed the activation of NF-κB and activating protein-1, as well as phosphorylation of p38 mitogen-activated protein kinase by electrophoretic mobility shift assay with super shift and/or Western blot. RESULTS: During and after cardiac surgery, animals subjected to hypothermia exhibited lower expression of TNF-α and cyclo-oxygenase-2 but not of inducible nitric oxide synthase. This was associated with lower activation of p38 mitogen-activated protein kinase and of its downstream effector activating protein-1 in hypothermic animals. In contrast, NF-κB activity was no different between groups. CONCLUSION: These findings indicate that the repression of TNF-α associated with moderate hypothermia during cardiac surgery is associated with inhibition of the mitogen-activated protein kinase p38/activating protein-1 pathway and not with inhibition of NF-κB. The use of moderate hypothermia during cardiac surgery may mitigate the perioperative systemic inflammatory response and its complications

Identification of a functional 3′,5′-cyclic adenosine monophosphate response element within the second promoter of the mouse somatostatin receptor type 2 gene

AbstractImportant physiological actions of somatostatin are mediated by the somatostatin receptor type 2. Its transcription is regulated by three tissue specific, alternative promoters. It is known that the mRNA of the somatostatin receptor type 2 gene is induced by cAMP, but little is known about the mechanisms underlying this regulation. We have identified and characterized a cAMP responsive element located at nucleotide −162 on the second promoter of the gene consisting of the classical palindromic octameric sequence 5′-TGACGTCA-3′. Using transient expression of reporter gene deletion constructs in NG108-15 cells the necessity of the intact element for forskolin-induced reporter gene activity was demonstrated. The first and the third promoter are not responsive to forskolin, nor did any promoter respond to the phorbol ester PMA. Electrophoretic mobility shift assays in combination with competition experiments suggest the interaction of the promoter element with the cAMP responsive element binding protein

PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Background: In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results: The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion: The present study highlights the existence of an inter-donor variability of expression of neuralrelated markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies

Textile Design of an Intervertebral Disc Replacement Device from Silk Yarn

Low back pain is often due to degeneration of the intervertebral discs (IVD). It is one of the most common age- and work-related problems in today’s society. Current treatments are not able to efficiently restore the full function of the IVD. Therefore, the aim of the present work was to reconstruct the two parts of the intervertebral disc—the annulus fibrosus (AF) and the nucleus pulposus (NP)—in such a way that the natural structural features were mimicked by a textile design. Silk was selected as the biomaterial for realization of a textile IVD because of its cytocompatibility, biodegradability, high strength, stiffness, and toughness, both in tension and compression. Therefore, an embroidered structure made of silk yarn was developed that reproduces the alternating fiber structure of +30° and −30° fiber orientation found in the AF and mimics its lamellar structure. The developed embroidered ribbons showed a tensile strength that corresponded to that of the natural AF. Fiber additive manufacturing with 1 mm silk staple fibers was used to replicate the fiber network of the NP and generate an open porous textile 3D structure that may serve as a reinforcement structure for the gel-like NP

Применение детандер-генератора для повышения ресурсоэффективности газораспределительной станции магистрального газопровода

Объектом исследования является: Газораспределительная станция "Урожай-20" магистрального газопровода "П-К" в Томской области. Цель работы: Выбор наиболее оптимального детандер–генераторного агрегата для использования потенциала природного газа при редуцировании на газораспределительной станции. В процессе исследования были проведены: аналитический обзор детандер–генераторных агрегатов применимых для газораспределительных станций различной производительности, изучение характеристик и специфики эксплуатации газораспределительной станции; выбор оптимального детандер–генераторного агрегата для использования его на ГРС-Ч "Урожай-20; проведение расчетов мощности и рентабельности применения детандер–генераторного агрегата.Object of the study: gas distribution station "Urozhay-20" of the main gas pipeline "P-K" in the of the Tomsk region. Purpose – Selection of the most optimal expander-generator set for using natural gas capabilities while reducing at a gas distribution station. The study included: analytical review of expander-generator sets applicable for gas distribution stations of various capacities, study of characteristics and specific features of operation of the gas distribution station, selection of the optimal expander-generator set for its use on GDS "Urozhai-20, carrying out of calculations of capacity and profitability of application of the expander-generator set, adoption of the most acceptable schemes for the introduction of an expander-generator set at AGRS "Urozhay-20"