Maintained mitochondrial integrity without oxygen in the anoxia tolerant crucian carp.

Very few vertebrates survive without oxygen (anoxia) for more than a few minutes. The crucian carp (Carassius carassius) survive months of anoxia at low temperatures and we hypothesised that they maintain mitochondrial membrane potential and function. Isolated crucian carp cardiomyocytes indeed maintained mitochondrial membrane potential after blocking complex-IV of the electron transport system with cyanide, while those of anoxia-intolerant trout depolarized. When complexes I-III were inhibited, crucian carp mitochondria depolarized, indicating that these complexes need to function during anoxia. Mitochondrial membrane potential depended on reversal of ATP synthase in chemical anoxia, as blocking with cyanide combined with oligomycin to inhibit ATP-synthase lead to depolarization. ATP-synthase activity was reduced in the heart after one week of anoxia in crucian carp, together with a downregulation of ATP-synthase subunit gene expression. However, the morphology of cardiac mitochondria were not affected by one-week anoxia, even with a large increase in mitofusin-2 expression. Cardiac citrate synthase activity was not affected by anoxia, while cytochrome-C oxidase activity was increased. We show how mitochondria respond to anoxia. A mechanistic understanding of how mitochondrial function can be maintained in anoxia may provide new perspectives to reduce mitochondrial damage in anoxia-sensitive organisms.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells.

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = -0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

When Our Best Friend Becomes Our Worst Enemy: The Mitochondrion in Trauma, Surgery, and Critical Illness.

Common for major surgery, multitrauma, sepsis, and critical illness, is a whole-body inflammation. Tissue injury is able to trigger a generalized inflammatory reaction. Cell death causes release of endogenous structures termed damage associated molecular patterns (DAMPs) that initiate a sterile inflammation. Mitochondria are evolutionary endosymbionts originating from bacteria, containing molecular patterns similar to bacteria. These molecular patterns are termed mitochondrial DAMPs (mDAMPs). Mitochondrial debris released into the extracellular space or into the circulation is immunogenic and damaging secondary to activation of the innate immune system. In the circulation, released mDAMPS are either free or exist in extracellular vesicles, being able to act on every organ and cell in the body. However, the role of mDAMPs in trauma and critical care is not fully clarified. There is a complete lack of knowledge how they may be counteracted in patients. Among mDAMPs are mitochondrial DNA, cardiolipin, N-formyl peptides, cytochrome C, adenosine triphosphate, reactive oxygen species, succinate, and mitochondrial transcription factor A. In this overview, we present the different mDAMPs, their function, release, targets, and inflammatory potential. In light of present knowledge, the role of mDAMPs in the pathophysiology of major surgery and trauma as well as sepsis, and critical care is discussed.

No knowledge gap in human physiology after remote teaching for second year medical students throughout the Covid-19 pandemic.

The COVID-19 pandemic had a disruptive effect on higher education. A critical question is whether these changes affected students' learning outcomes. Knowledge gaps have consequences for future learning and may-in health professionals' education-also pose a threat to patient safety. Current research has shortcomings and does not allow for clear-cut interpretation. Our context is instruction in human physiology in an undergraduate medical program from high stakes end of term examinations. The sequence of imposed measures to slow the COVID-19 pandemic created a natural experiment, allowing for comparisons in performance during in-person versus remote instruction.In a two-factorial design, mode of instruction (in-person vs. remote) and mode of assessment (in-person vs. remote) were analyzed using both basic (non-parametric statistics, T-tests) and advanced statistical methods (linear mixed-effects model; resampling techniques). Test results from a total of N = 1095 s-year medical students were included in the study.We did not find empirical evidence of knowledge gaps; rather, students received comparable or higher scores during remote teaching. We interpret these findings as empirical evidence that both students and teachers adapted to pandemic disruption in a way that did not lead to knowledge gaps.We conclude that highly motivated students had no reduction in academic achievement. Moreover, we have developed an accessible digital exam system for secure, fair, and effective assessments which is sufficiently defensible for making pass/fail decisions.

Energy substrate metabolism, mitochondrial structure and oxidative stress after cardiac ischemia-reperfusion in mice lacking UCP3.

Myocardial ischemia-reperfusion (IR) injury may result in cardiomyocyte dysfunction. Mitochondria play a critical role in cardiomyocyte recovery after IR injury. The mitochondrial uncoupling protein 3 (UCP3) has been proposed to reduce mitochondrial reactive oxygen species (ROS) production and to facilitate fatty acid oxidation. As both mechanisms might be protective following IR injury, we investigated functional, mitochondrial structural, and metabolic cardiac remodeling in wild-type mice and in mice lacking UCP3 (UCP3-KO) after IR. Results showed that infarct size in isolated perfused hearts subjected to IR ex vivo was larger in adult and old UCP3-KO mice than in equivalent wild-type mice, and was accompanied by higher levels of creatine kinase in the effluent and by more pronounced mitochondrial structural changes. The greater myocardial damage in UCP3-KO hearts was confirmed in vivo after coronary artery occlusion followed by reperfusion. S1QEL, a suppressor of superoxide generation from site IQ in complex I, limited infarct size in UCP3-KO hearts, pointing to exacerbated superoxide production as a possible cause of the damage. Metabolomics analysis of isolated perfused hearts confirmed the reported accumulation of succinate, xanthine and hypoxanthine during ischemia, and a shift to anaerobic glucose utilization, which all recovered upon reoxygenation. The metabolic response to ischemia and IR was similar in UCP3-KO and wild-type hearts, being lipid and energy metabolism the most affected pathways. Fatty acid oxidation and complex I (but not complex II) activity were equally impaired after IR. Overall, our results indicate that UCP3 deficiency promotes enhanced superoxide generation and mitochondrial structural changes that increase the vulnerability of the myocardium to IR injury.

Mitochondria-derived damage-associated molecular patterns and inflammation in the ischemic-reperfused heart.

Cardiac cell death after myocardial infarction release endogenous structures termed damage-associated molecular patterns (DAMPs) that trigger the innate immune system and initiate a sterile inflammation in the myocardium. Cardiomyocytes are energy demanding cells and 30% of their volume are mitochondria. Mitochondria are evolutionary endosymbionts originating from bacteria containing molecular patterns similar to bacteria, termed mitochondrial DAMPs (mDAMPs). Consequently, mitochondrial debris may be particularly immunogenic and damaging. However, the role of mDAMPs in myocardial infarction is not clarified. Identifying the most harmful mDAMPs and inhibiting their early inflammatory signaling may reduce infarct size and the risk of developing post-infarct heart failure. The focus of this review is the role of mDAMPs in the immediate pro-inflammatory phase after myocardial infarction before arrival of immune cells in the myocardium. We discuss different mDAMPs, their role in physiology and present knowledge regarding their role in the inflammatory response of acute myocardial infarction.

Toll-like receptor 9 signaling after myocardial infarction: Role of p66ShcA adaptor protein.

During myocardial infarction, cellular debris is released, causing a sterile inflammation via pattern recognition receptors. These reactions amplify damage and promotes secondary heart failure. The pattern recognition receptor, Toll-like receptor 9 (TLR9) detects immunogenic fragments of endogenous DNA, inducing inflammation by NFκB. The p66ShcA adaptor protein plays an important role in both ischemic myocardial damage and immune responses. We hypothesized that p66ShcA adaptor protein promotes DNA-sensing signaling via the TLR9 pathway after myocardial infarction. TLR9 protein expression increased in cardiac tissue from patients with end-stage heart failure due to ischemic heart disease. Myocardial ischemia in mice in vivo induced gene expression of key TLR9 pathway proteins (MyD88 and Unc93b1). In this model, a functional link between TLR9 and p66ShcA was revealed as; (i) ischemia-induced upregulation of TLR9 protein was abrogated in myocardium of p66ShcA knockout mice; (ii) when p66ShcA was overexpressed in NFkB reporter cells stably expressing TLR9, NFkB-activation increased during stimulation with the TLR9 agonist CpG B; (iii) in cardiac fibroblasts, p66ShcA overexpression caused TLR9 upregulation. Co-immunoprecipitation showed that ShcA proteins and TLR9 may be found in the same protein complex, which was dissipated upon TLR9 stimulation in vivo. A proximity assay confirmed the co-localization of TLR9 and ShcA proteins. The systemic immune response after myocardial ischemia was dampened in p66ShcA knockout mice as interleukin-4, -17 and -22 expression in mononuclear cells isolated from spleens was reduced. In conclusion, p66ShcA adaptor may be an interaction partner and a regulator of the TLR9 pathway post-infarction.

Multi-omics of in vitro aortic valve calcification.

Heart valve calcification is an active cellular and molecular process that partly remains unknown. Osteogenic differentiation of valve interstitial cells (VIC) is a central mechanism in calcific aortic valve disease (CAVD). Studying mechanisms in CAVD progression is clearly needed. In this study, we compared molecular mechanisms of osteogenic differentiation of human VIC isolated from healthy donors or patients with CAVD by RNA-seq transcriptomics in early timepoint (48 h) and by shotgun proteomics at later timepoint (10th day). Bioinformatic analysis revealed genes and pathways involved in the regulation of VIC osteogenic differentiation. We found a high amount of stage-specific differentially expressed genes and good accordance between transcriptomic and proteomic data. Functional annotation of differentially expressed proteins revealed that osteogenic differentiation of VIC involved many signaling cascades such as: PI3K-Akt, MAPK, Ras, TNF signaling pathways. Wnt, FoxO, and HIF-1 signaling pathways were modulated only at the early timepoint and thus probably involved in the commitment of VIC to osteogenic differentiation. We also observed a significant shift of some metabolic pathways in the early stage of VIC osteogenic differentiation. Lentiviral overexpression of one of the most upregulated genes (ZBTB16, PLZF) increased calcification of VIC after osteogenic stimulation. Analysis with qPCR and shotgun proteomics suggested a proosteogenic role of ZBTB16 in the early stages of osteogenic differentiation.

Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview.

Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo, in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.

Early cardiac-chamber-specific fingerprints in heart failure with preserved ejection fraction detected by FTIR and Raman spectroscopic techniques.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) is a matter of investigation and its diagnosis remains challenging. Although the mechanisms that are responsible for the development of HFpEF are not fully understood, it is well known that nearly 80% of patients with HFpEF have concomitant hypertension. We investigated whether early biochemical alterations were detectable during HFpEF progression in salt-induced hypertensive rats, using Fourier-transformed infrared (FTIR) and Raman spectroscopic techniques as a new diagnostic approach. Greater protein content and, specifically, greater collagen deposition were observed in the left atrium and right ventricle of hypertensive rats, together with altered metabolism of myocytes. Additionally, Raman spectra indicated a conformational change, or different degree of phosphorylation/methylation, in tyrosine-rich proteins. A correlation was found between tyrosine content and cardiac fibrosis of both right and left ventricles. Microcalcifications were detected in the left and right atria of control animals, with a progressive augmentation from six to 22 weeks. A further increase occurred in the left ventricle and right atrium of 22-week salt-fed animals, and a positive correlation was shown between the mineral deposits and the cardiac size of the left ventricle. Overall, FTIR and Raman techniques proved to be sensitive to early biochemical changes in HFpEF and preceded clinical humoral and imaging markers.

Extracellular histones are a target in myocardial ischaemia-reperfusion injury.

AIMS: Acute myocardial infarction causes lethal cardiomyocyte injury during ischaemia and reperfusion (I/R). Histones have been described as important Danger Associated Molecular Proteins (DAMPs) in sepsis. The objective of this study was to establish whether extracellular histone release contributes to myocardial infarction. METHODS AND RESULTS: Isolated, perfused rat hearts were subject to I/R. Nucleosomes and histone-H4 release was detected early during reperfusion. Sodium-ß-O-Methyl cellobioside sulfate (mCBS), a newly developed histone-neutralizing compound, significantly reduced infarct size whilst also reducing the detectable levels of histones. Histones were directly toxic to primary adult rat cardiomyocytes in vitro. This was prevented by mCBS or HIPe, a recently described, histone-H4 neutralizing peptide, but not by an inhibitor of TLR4, a receptor previously reported to be involved in DAMP-mediated cytotoxicity. Furthermore, TLR4-reporter HEK293 cells revealed that cytotoxicity of histone H4 was independent of TLR4 and NF-κB. In an in vivo rat model of I/R, HIPe significantly reduced infarct size. CONCLUSION: Histones released from the myocardium are cytotoxic to cardiomyocytes, via a TLR4-independent mechanism. The targeting of extracellular histones provides a novel opportunity to limit cardiomyocyte death during I/R injury of the myocardium.

Isolated Plin5-deficient cardiomyocytes store less lipid droplets than normal, but without increased sensitivity to hypoxia.

Plin5 is abundantly expressed in the heart where it binds to lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5-/- mice to study the role of Plin5 for fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia. Cardiomyocytes isolated from Plin5-/- mice cultured with oleic acid stored less LDs than Plin5+/+, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/- cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia compared to Plin5+/+. In isolated, perfused hearts, deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight, and apart from modest elevated expression of mRNAs for beta myosin heavy chain Myh7 and the fatty acid transporter Cd36, other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content of isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in mouse models during myocardial ischemia.

Mitochondrial and mitochondrial-independent pathways of myocardial cell death during ischaemia and reperfusion injury.

Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial-independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled 'Mitochondria as targets of acute cardioprotection' and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.

A Novel Ex Vivo Model of Aortic Valve Calcification. A Preliminary Report.

Background: No pharmacological treatment exists to prevent or stop the calcification process of aortic valves causing aortic stenosis. The aim of this study was to develop a robust model of induced calcification in whole aortic valve leaflets which could be suitable for studies of the basic mechanisms and for testing potentially inhibitory drugs. Methods: Pig hearts were obtained from a commercial abattoir. The aortic valve leaflets were dissected free and randomized between experimental groups. Whole leaflets were cultured in individual wells. Two growth media were used for cultivation: standard growth medium and an antimyofibroblastic growth medium. The latter was employed to inhibit contraction of the leaflet into a ball-like structure. Calcification was induced in the growth medium by supplementation with an osteogenic medium. Leaflets were cultivated for four weeks and medium was changed every third day. To block calcification, the inhibitor SNF472 (a formulation of the hexasodium salt of myo-inositol hexaphosphate hexasodium salt) was used at concentrations between 1 and 100 µM. After cultivation for four weeks the leaflets were snap frozen in liquid nitrogen and kept at -80 °C until blind assessment of the calcium amount in leaflets by inductively coupled plasma optical emission spectroscopy. For statistical analysis, a Kruskal-Wallis test with Dunn's post-test was applied. Results: Osteodifferentiation with calcium accumulation was in principle absent when standard medium was used. However, when the antimyofibroblastic medium was used, a strong calcium accumulation was induced (p = 0.006 compared to controls), and this was blocked in a dose-dependent manner by the calcification inhibitor SNF472 (p = 0.008), with an EC50 of 3.3 µM. Conclusion: A model of experimentally induced calcification in cultured whole leaflets from porcine aortic valves was developed. This model can be useful for studying the basic mechanisms of valve calcification and to test pharmacological approaches to inhibit calcification.

Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties.

Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and reoxygenation.

BACKGROUND AND PURPOSE: Cellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage-associated-molecular-pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll-like receptor 9 (TLR9). EXPERIMENTAL APPROACH: We hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia-reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA. KEY RESULTS: Expression of the pro-inflammatory cytokines IL-1ß and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia-reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL-1ß/TNFα and the nucleolin inhibitor AS1411 reduced interleukin-6 release in adult mouse cardiomyocytes. mtDNA bound 10-fold stronger than nDNA to nucleolin. In HEK293-NF-κB reporter cells, mtDNA induced NF-κB activity in normoxia, while CpG-DNA and hypoxia-reoxygenation, synergistically induced TLR9-dependent NF-κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG-DNA. Inhibition of endocytosis did not reduce CpG-DNA uptake in cardiomyocytes. CONCLUSION AND IMPLICATIONS: mtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia-reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.

Mammalian Target of Rapamycin (mTOR) and the Proteasome Attenuates IL-1ß Expression in Primary Mouse Cardiac Fibroblasts.

Background: IL-1ß is a highly potent pro-inflammatory cytokine and its secretion is tightly regulated. Inactive pro-IL-1ß is transcribed in response to innate immune receptors activating NFκB. If tissue damage occurs, danger signals released from necrotic cells, such as ATP, can activate NLRP3-inflammasomes (multiprotein complexes consisting of NLRP3, ASC, and active caspase-1) which cleaves and activates pro-IL-1ß. NLRP3 activation also depends on NEK7 and mitochondrial ROS-production. Thus, IL-1ß secretion may be regulated at the level of each involved component. We have previously shown that NLRP3-dependent IL-1ß release can be induced in cardiac fibroblasts by pro-inflammatory stimuli. However, anti-inflammatory mechanisms targeting IL-1ß release in cardiac cells have not been investigated. mTOR is a key regulator of protein metabolism, including autophagy and proteasome activity. In this study we explored whether autophagy or proteasomal degradation are regulators of NLRP3 inflammasome activation and IL-1ß release from cardiac fibroblasts. Methods and Results: Serum starvation selectively reduced LPS/ATP-induced IL-1ß secretion from cardiac fibroblasts. However, no other inflammasome components, nor mitochondrial mass, were affected. The mTOR inhibitor rapamycin restored pro-IL-1ß protein levels as well as LPS/ATP-induced IL-1ß release from serum starved cells. However, neither serum starvation nor rapamycin induced autophagy in cardiac fibroblasts. Conversely, chloroquine and bafilomycin A (inhibitors of autophagy) and betulinic acid (a proteasome activator) effectively reduced LPS-induced pro-IL-1ß protein levels. Key findings were reinvestigated in human monocyte-derived macrophages. Conclusion: In cardiac fibroblasts, mTOR inhibition selectively favors pro-IL-1ß synthesis while proteasomal degradation and not autophagy is the major catabolic anti-inflammatory mechanism for degradation of this cytokine.

Mechanical stress alters the expression of calcification-related genes in vascular interstitial and endothelial cells.

OBJECTIVES: Vascular wall calcification is a major pathophysiological component of atherosclerotic disease with many similarities to osteogenesis. Mechanical stress of the vascular wall may theoretically contribute to the proliferative processes by endothelial and interstitial cells. The aim of the study was to investigate the effect of mechanical stress on the expression of some calcification-related genes in primary human endothelial and interstitial cells, and how endothelial cells may stimulate the fibroblast and smooth muscle cells. METHODS: Human umbilical vein endothelial and interstitial cells were subjected to cyclic stretch using a FlexCell® bioreactor, and interstitial cells were also subjected to tensile strain in cultures embedded in 3-dimensional collagen gels. The medium from endothelial cells was used to stimulate the gel-cultured interstitial cells, or the endothelium was sown directly on top. For comparison, human endothelial and smooth muscle cells were isolated from aortic wall fragments of patients with and without the aortic aneurysm. The expression of genes was measured using quantitative PCR. RESULTS: Four hours of cyclic stretch applied to cultured endothelial cells upregulated the mRNA expression of bone morphogenetic protein 2 (BMP-2), a major procalcific growth factor. When applied to a 3-dimensional culture of vascular interstitial cells, the medium from prestretched endothelial cells decreased the expression of BMP-2 and periostin mRNA in the fibroblasts. The static tension in gel-cultured interstitial cells upregulated BMP-2 mRNA expression. The addition of endothelial cells on the top of this culture also reduced mRNA of anticalcific genes, periostin and osteopontin. Similar changes were observed in smooth muscle cells from human aortic aneurysms compared to cells from the healthy aorta. Aortic aneurysm endothelial cells also showed an increased expression of BMP-2 mRNA. CONCLUSIONS: Endothelial cells respond to mechanical stress by upregulation of pro-osteogenic factor BMP-2 mRNA and modulate the expression of other osteogenic factors in vascular interstitial cells. Endothelial cells may, thus, contribute to vascular calcification when exposed to mechanical stress.

Release of Mitochondrial and Nuclear DNA During On-Pump Heart Surgery: Kinetics and Relation to Extracellular Vesicles.

During heart surgery with cardiopulmonary bypass (CPB), the release of mitochondrial (mtDNA) and nuclear DNA (nDNA) and their association to extracellular vesicles were investigated. In patients undergoing elective coronary artery bypass grafting (CABG, n = 12), blood was sampled before, during, and after surgery from peripheral artery, pulmonary artery, and the coronary sinus. Plasma was separated in three fractions: microvesicles, exosomes, and supernatant. mtDNA and nDNA were measured by qPCR. mtDNA and nDNA levels increased after start of surgery, but before CPB, and increased further during CPB. mtDNA copy number was about 1000-fold higher than nDNA. mtDNA was predominantly localized to the vesicular fractions in plasma, whereas nDNA was predominantly in the supernatant. The amount of free mtDNA increased after surgery. There was no net release or disappearance of DNAs across the pulmonary, systemic, or coronary circulation. Extracellular DNAs, in particular mtDNA, may be important contributors to the whole-body inflammation during CPB.

Inflammation and Mechanical Stress Stimulate Osteogenic Differentiation of Human Aortic Valve Interstitial Cells.

Background: Aortic valve calcification is an active proliferative process, where interstitial cells of the valve transform into either myofibroblasts or osteoblast-like cells causing valve deformation, thickening of cusps and finally stenosis. This process may be triggered by several factors including inflammation, mechanical stress or interaction of cells with certain components of extracellular matrix. The matrix is different on the two sides of the valve leaflets. We hypothesize that inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells (VICs) and this may depend on the side of the leaflet. Methods: Interstitial cells isolated from healthy and calcified human aortic valves were cultured on collagen or elastin coated plates with flexible bottoms, simulating the matrix on the aortic and ventricular side of the valve leaflets, respectively. The cells were subjected to 10% stretch at 1 Hz (FlexCell bioreactor) or treated with 0.1 µg/ml lipopolysaccharide, or both during 24 h. Gene expression of myofibroblast- and osteoblast-specific genes was analyzed by qPCR. VICs cultured in presence of osteogenic medium together with lipopolysaccharide, 10% stretch or both for 14 days were stained for calcification using Alizarin Red. Results: Treatment with lipopolysaccharide increased expression of osteogenic gene bone morphogenetic protein 2 (BMP2) (5-fold increase from control; p = 0.02) and decreased expression of mRNA of myofibroblastic markers: α-smooth muscle actin (ACTA2) (50% reduction from control; p = 0.0006) and calponin (CNN1) (80% reduction from control; p = 0.0001) when cells from calcified valves were cultured on collagen, but not on elastin. Mechanical stretch of VICs cultured on collagen augmented the effect of lipopolysaccharide. Expression of periostin (POSTN) was inhibited in cells from calcified donors after treatment with lipopolysaccharide on collagen (70% reduction from control, p = 0.001), but not on elastin. Lipopolysaccharide and stretch both enhanced the pro-calcific effect of osteogenic medium, further increasing the effect when combined for cells cultured on collagen, but not on elastin. Conclusion: Inflammation and mechanical stress trigger expression of osteogenic genes in VICs in a side-specific manner, while inhibiting the myofibroblastic pathway. Stretch and lipopolysaccharide synergistically increase calcification.