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.

Circulating MicroRNA-210 Concentrations in Patients with Acute Heart Failure: Data from the Akershus Cardiac Examination 2 Study.

BACKGROUND: MicroRNA (miR)-210 expression is induced by acute and chronic hypoxia and provides prognostic information in patients with aortic stenosis and acute coronary syndrome. We hypothesized that circulating miR-210 concentrations could provide diagnostic and prognostic information in patients with acute heart failure (HF). METHODS: We measured miR-210 concentrations in serum samples on admission from 314 patients hospitalized for acute dyspnea and 9 healthy control subjects. The diagnostic and prognostic properties of miR-210 were tested in patients after adjudication of all diagnoses and with median follow-up of 464 days. RESULTS: All patients and control subjects had miR-210 concentrations within the range of detection, and the analytical variation was low as the coefficient of variation of synthetic spike-in RNA was 4%. Circulating miR-210 concentrations were increased in patients with HF compared to healthy control subjects, but miR-210 concentrations did not separate patients with acute HF (n = 143) from patients with non-HF-related dyspnea (n = 171): the area under the curve was 0.50 (95% CI 0.43-0.57). Circulating miR-210 concentrations were associated with mortality (n = 114) after adjustment for clinical risk factors (hazard ratio 1.65 [95% CI 1.03-2.62] per unit miR-210 increase), but this association was attenuated and not significant after adjustment for established cardiac protein biomarkers. CONCLUSIONS: Circulating miR-210 concentrations are associated with mortality, but do not add to established protein biomarkers for diagnosis or prognosis in patients with acute dyspnea.

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.

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.

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.

Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

Osteoblast Differentiation at a Glance.

Ossification is a tightly regulated process, performed by specialized cells called osteoblasts. Dysregulation of this process may cause inadequate or excessive mineralization of bones or ectopic calcification, all of which have grave consequences for human health. Understanding osteoblast biology may help to treat diseases such as osteogenesis imperfecta, calcific heart valve disease, osteoporosis, and many others. Osteoblasts are bone-building cells of mesenchymal origin; they differentiate from mesenchymal progenitors, either directly or via an osteochondroprogenitor. The direct pathway is typical for intramembranous ossification of the skull and clavicles, while the latter is a hallmark of endochondral ossification of the axial skeleton and limbs. The pathways merge at the level of preosteoblasts, which progress through 3 stages: proliferation, matrix maturation, and mineralization. Osteoblasts can also differentiate into osteocytes, which are stellate cells populating narrow interconnecting passages within the bone matrix. The key molecular switch in the commitment of mesenchymal progenitors to osteoblast lineage is the transcription factor cbfa/runx2, which has multiple upstream regulators and a wide variety of targets. Upstream is the Wnt/Notch system, Sox9, Msx2, and hedgehog signaling. Cofactors of Runx2 include Osx, Atf4, and others. A few paracrine and endocrine factors serve as coactivators, in particular, bone morphogenetic proteins and parathyroid hormone. The process is further fine-tuned by vitamin D and histone deacetylases. Osteoblast differentiation is subject to regulation by physical stimuli to ensure the formation of bone adequate for structural and dynamic support of the body. Here, we provide a brief description of the various stimuli that influence osteogenesis: shear stress, compression, stretch, micro- and macrogravity, and ultrasound. A complex understanding of factors necessary for osteoblast differentiation paves a way to introduction of artificial bone matrices.

Serglycin in Quiescent and Proliferating Primary Endothelial Cells.

Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1ß induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1ß activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development.

Higher TNFα responses in young males compared to females are associated with attenuation of monocyte adenylyl cyclase expression.

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation. Males and females, age groups 20-30 years and 50-70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6h, p=0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p=0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases.

Deletion of the aquaporin-4 gene alters expression and phosphorylation of protective kinases in the mouse heart.

AIM: Aquaporins are channel-forming proteins highly permeable to water and some small molecular solutes. We have previously shown that aquaporin-4 knockout mice have increased tolerance to ischemia. However, the mechanism of cardioprotection was unclear. The aim of the current study was to investigate the effects of aquaporin-4 deletion on baseline expression and phosphorylation of some cardioprotective protein kinases. METHODS: Proteins were extracted from hearts of aquaporin-4 knockout mice and littermate wild-type controls and analyzed with Western blot. Samples were taken from young (≤ 6 months of age), and old (≥ 1 year) mice. RESULTS: Western blots showed three different isoforms of aquaporin-4 in wild types, likely representing M1, M23, and Mz. Total AMP-dependent kinase expression was decreased in aquaporin-4 knockout hearts by 18 ± 13% (p = 0.02), while the expression of Akt kinase, extracellular signal regulated kinase 1/2, protein kinase C-epsilon, mitogen-associated kinase P38 and C-Jun N-terminal kinase was unchanged. The phosphorylation of Akt kinase was reduced in hearts from knockout mice by 41 ± 16% (p = 0.01). No other alterations in phosphorylation were found. These effects were only detected in young mice. CONCLUSION: Deletion of the aquaporin-4 gene decreased AMP-dependent kinase expression and Akt kinase phosphorylation in the heart. These changes may influence energy metabolism and endogenous cardioprotection.

Expression of bone morphogenetic protein 4 and its receptors in the remodeling heart.

AIMS: Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure. MAIN METHODS: Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n=15) or ischemic heart disease (CAD; n=9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n=7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n=11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n=7 at each time point). KEY FINDINGS: Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H2O2 injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia-reoxygenation injury. SIGNIFICANCE: Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.

Cardiac aquaporins.

Aquaporins are a group of proteins with high-selective permeability for water. A subgroup called aquaglyceroporins is also permeable to glycerol, urea and a few other solutes. Aquaporin function has mainly been studied in the brain, kidney, glands and skeletal muscle, while the information about aquaporins in the heart is still scarce. The current review explores the recent advances in this field, bringing aquaporins into focus in the context of myocardial ischemia, reperfusion, and blood osmolarity disturbances. Since the amount of data on aquaporins in the heart is still limited, examples and comparisons from better-studied areas of aquaporin biology have been used. The human heart expresses aquaporin-1, -3, -4 and -7 at the protein level. The potential roles of aquaporins in the heart are discussed, and some general phenomena that the myocardial aquaporins share with aquaporins in other organs are elaborated. Cardiac aquaporin-1 is mostly distributed in the microvasculature. Its main role is transcellular water flux across the endothelial membranes. Aquaporin-4 is expressed in myocytes, both in cardiac and in skeletal muscle. In addition to water flux, its function is connected to the calcium signaling machinery. It may play a role in ischemia-reperfusion injury. Aquaglyceroporins, especially aquaporin-7, may serve as a novel pathway for nutrient delivery into the heart. They also mediate toxicity of various poisons. Aquaporins cannot influence permeability by gating, therefore, their function is regulated by changes of expression-on the levels of transcription, translation (by microRNAs), post-translational modification, membrane trafficking, ubiquitination and subsequent degradation. Studies using mice genetically deficient for aquaporins have shown rather modest changes in the heart. However, they might still prove to be attractive targets for therapy directed to reduce myocardial edema and injury caused by ischemia and reperfusion.

Aquaporin-1 in cardiac endothelial cells is downregulated in ischemia, hypoxia and cardioplegia.

Aquaporin-1 (AQP1) is expressed in human and mouse hearts, but little is known about its cellular and subcellular localization and regulation. The aim of this study was to investigate the localization of AQP1 in the mouse heart and to determine the effects of ischemia and hypoxia on its expression. Mouse myocardial cells were freshly isolated and split into cardiomyocyte and non-cardiomyocyte fractions. Isolated, Langendorff-perfused C57Bl6 mouse hearts (n=46) were harvested with no intervention, subjected to 35min of ischemia or ischemia followed by 60min of reperfusion. Eleven mouse hearts were perfusion-fixed for electron microscopy. Forty C57Bl6 mice were exposed to normobaric hypoxia for one or two weeks (n=12). Needle biopsies of human left ventricular myocardium were sampled (n=30) during coronary artery bypass surgery before cardioplegia and after 30min of reperfusion. Human umbilical vein endothelial cells (HUVECs) were subjected to 4h of hypoxia with reoxygenation for either 4 or 24h. AQP1 expression was studied by electron microscopy with immunogold labeling, Western blot, and qPCR. Expression of miR-214 and miR-320 in HUVECs with hypoxia was studied with qPCR. HUVECs were then transfected with precursors and inhibitors of miR-214. AQP1 expression was confined to cardiac endothelial cells, with no signal in cardiomyocytes or cardiac fibroblasts. Immunogold electron microscopy showed AQP1 expression in endothelial caveolae with equal distribution along the basal and apical membranes. Ischemia and reperfusion tended to decrease AQP1 mRNA expression in mouse hearts by 37±9% (p=0.06), while glycosylated AQP1 protein was reduced by 16±9% (p=0.03). No difference in expression was found between ischemia alone and ischemia-reperfusion. In human left ventricles AQP1 mRNA expression was reduced following cardioplegia and reperfusion (p=0.008). Hypoxia in mice reduced AQP1 mRNA expression by 20±7% (p<0.0001), as well as both glycosylated (-47±10%, p=0.03) and glycan-free protein (-34±16%, p=0.05). Hypoxia and reoxygenation in HUVECs downregulated glycan-free AQP1 protein (-34±24%, p=0.04) and upregulated miR-214 (+287±52%, p<0.05). HUVECs transfected with anti-miR-214 had increased glycosylated (1.5 fold) and glycan-free (2 fold) AQP1. AQP1 in mouse hearts is localized to endothelial cell membranes and caveolae. Cardioplegia, ischemia and hypoxia decrease AQP1 mRNA as well as total protein expression and glycosylation, possibly regulated by miR-214.

Transient hyperosmolality modulates expression of cardiac aquaporins.

PURPOSE: Hyperosmolarity is a common complication in intensive care patients, dysregulating water balance in many organs including brain and heart. The aquaporin (AQP) water channels, in particular AQP1 and -4, have been suggested to play an important role in fluid homeostasis of the myocardium. In many organs AQP expression is regulated by osmolarity, drastically altering water permeability of the cell membranes. The aim of our study was to investigate if plasma hyperosmolality may regulate cardiac expression of AQP1 and -4, and if so, at which magnitude and time frame such regulation takes place. METHODS: C57Bl6 mice were injected intraperitoneally with either 1.5 ml 0.154 Mol (isoosmotic), 0.5 ml 1 Mol (mild hyperosmotic) or 0.5 ml 2 Mol (strong hyperosmotic) NaCl. Plasma, hearts, and forebrains were harvested before injection ("time 0"), and after 1, 4, 8 and 24 h. AQP1 and -4 expression were analyzed using qPCR and Western blot. RESULTS: Isoosmotic and mild hyperosmotic injections caused no important changes in cardiac AQP expression. Strong hyperosmotic NaCl injections induced an upregulation of AQP1 mRNA and glycosylated fraction of AQP1 protein in the heart without changes of the total protein. AQP4 mRNA and protein decreased in the heart and increased in the brain after hyperosmotic NaCl. The change in AQP4 protein content in the brain preceded the increase of mRNA. CONCLUSION: As in the brain, expression of AQP1 and -4 in the heart is influenced by changes in plasma osmolality. Changes in AQP expression may alter cardiac function in hyperosmotic states.

Aquaporin-4 in the heart: expression, regulation and functional role in ischemia.

Aquaporins (AQPs) are channel-forming membrane proteins highly permeable to water. AQP4 is found in mammalian hearts; however, its expression sites, regulation and function are largely unknown. The aim was to investigate cardiac AQP4 expression in humans and mice, its regulation by ischemia and hypoxia, and in particular its role in cardiac ischemic injury using AQP4 knockout (KO) mice. Comparable levels of AQP4 were detected by Western blot and qPCR in biopsies from human donor hearts and wild type C57Bl6 mouse hearts. In mice, AQP4 was expressed on cardiomyocyte plasmalemma (qPCR, Western blot, immunogold), and its mRNA decreased following ischemia/reperfusion (isolated hearts, p = 0.02) and after normobaric hypoxia in vivo (oxygen fraction 10 % for 1 week, p < 0.001). Isolated hearts from AQP4 KO mice undergoing global ischemia and reperfusion had reduced infarct size (p = 0.05) and attenuated left ventricular end-diastolic pressure during reperfusion (p = 0.04). Infarct size was also reduced in AQP4 KO mice 24 h after left coronary artery ligation in vivo (p = 0.036). AQP4 KO hearts had no compensatory change in AQP1 protein expression. AQP4 KO cardiomyocytes were partially resisted to hypoosmotic stress in the presence of hypercontracture. AQP4 is expressed in human and mouse hearts, in the latter confined to the cardiomyocyte plasmalemma. AQP4 mRNA expression is downregulated by hypoxia and ischemia. Deletion of AQP4 is protective in acute myocardial ischemia-reperfusion, and this molecule might be a future target in the treatment of acute myocardial infarction.

Hyperoxia during early reperfusion does not increase ischemia/reperfusion injury.

OBJECTIVE: Oxygen is routinely administered to patients undergoing acute myocardial infarction as well as during revascularization procedures and cardiac surgery. Because reactive oxygen species are mediators of ischemia/reperfusion injury, increased oxygen availability might theoretically aggravate myocardial injury during reperfusion. We hypothesized that ventilation with a hyperoxic gas at start of reperfusion might increase ischemia/reperfusion injury. METHODS: Rats were anesthetized with isoflurane and ventilated with 40% oxygen. The animals were subjected to 40 min of regional myocardial ischemia and 120 min of reperfusion. In the test group, rats (n=11) were ventilated with a normobaric hyperoxic gas (95% O2) during the last 10 min of ischemia and the first 10 min of reperfusion. Control rats (n=14) were ventilated with 40% O2 throughout the experiments. Due to irreversible reperfusion arrhythmias, one animal in the hyperoxia group and six animals in the control group were excluded. Hearts (n=8 in the control group and n=10 in the test group) were harvested for measurement of infarct size. RESULTS: The incidence of lethal arrhythmias was 1/11 in the test group and 6/14 in the control group (p=0.06). Reperfusion with normobaric hyperoxia did not influence infarct size (20±8% of area at risk) compared with the normoxia group (24±8% and of area at risk), respectively (mean±SD, p>0.2). CONCLUSION: Normobaric hyperoxia during early reperfusion did not increase ischemia/reperfusion injury.

Cecal ligation and puncture sepsis is associated with attenuated expression of adenylyl cyclase 9 and increased miR142-3p.

The host inflammatory response in sepsis may be resolved by endogenous anti-inflammatory immune cell responses, avoiding fatal pathogenesis, organ injury, and death. The intracellular signaling mediator cyclic 3'5'-adenosine monophosphate is a potent modulator of inflammatory responses and initiates the polarization of immune cells in a direction that suppresses inflammatory activation. Cyclic 3'5'-adenosine monophosphate is enzymatically produced by adenylyl cyclases (ACs). The expression of ACs is previously shown to be reduced in rat organs after in vivo endotoxemia, concurrent with the progressing systemic inflammation. In the present study, tissue AC gene expression and regulation are explored in a rat model of cecal ligation and puncture (CLP) sepsis. Eighteen hours after CLP operation, expression of several AC isoforms in the liver, spleen, and kidney was reduced, significantly so for AC9 in all tissues. AC9 expression is regulated by the microRNA miR142-3p in T cells. When microRNA was extracted and amplified for miR142-3p expression, it was increasingly expressed 18 h after CLP. A correlation between increased miR142-3p and decreased AC9 expression was found in the liver, kidney, and spleen, and when hepatocytes, Kupffer cells (KCs), and liver sinusoidal endothelial cells were isolated after CLP, reduced AC expression and increased miR142-3p expression were found in KCs and liver sinusoidal endothelial cells. Transfecting a miR142-3p inhibitor probe in rat KCs abolished LPS-mediated AC9 inhibition in vitro. These results indicate that CLP leads to miR142-3p-mediated AC9 reduction in liver macrophages, which may further limit cyclic 3'5'-adenosine monophosphate signaling and the ability of macrophages to resolve the proinflammatory response.

Inadvertent phosphorylation of survival kinases in isolated perfused hearts: a word of caution.

The isolated perfused heart is an important model in cardiovascular research. We hypothesized that the perfusion procedure per se will phosphorylate some protein kinases important in pre- and postconditioning. Isolated hearts were Langendorff-perfused for 20 min with or without an intraventricular balloon (rats and mice), or in the working heart mode (mice) and compared to non-perfused controls with respect to protein phosphorylation. Rat hearts were also perfused for 20 and 50 min in the Langendorff mode to investigate the effect of perfusion time on phosphorylation. Western blot analysis showed that perfusion per se induced a massive phosphorylation of ERK 1/2, P38-MAPK, JNK, AMPK, but decreased phosphorylation of AKT in the isolated rat and mouse heart. However, during ongoing perfusion the phosphorylation of these kinases was reduced. Langendorff-perfusion without the intraventricular balloon caused less phosphorylation of ERK 1/2, P38-MAPK and JNK, but had no effect on AMPK. In working hearts phosphorylation of kinases was similar to that of Langendorff-perfused hearts without the balloon. Our findings indicate that excising, handling and perfusion induce a time dependent phosphorylation of stress kinases. The presence of the intraventricular balloon caused the strongest phosphorylation, thus Langendorff-perfused hearts might be partly protected by the perfusion procedure if stress kinases are protective in pre- and postconditioning. This might explain conflicting results obtained with different models of both pre- and postconditioning, and the isolated heart might in some situations be suboptimal for such studies.