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.

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.

Microvascular remodeling following skin injury.

OBJECTIVE: The aim of this study was to describe possible remodeling (i.e., dilatation and elongation) of papillary capillaries induced by increased oxygen demand for the repair process following a skin wound. METHODS: Computer-assisted video microscopy was used to examine 10 healthy volunteers before (baseline) and after (≈1 h and ≈24 h) an incision (5 mm long and 1 mm deep) on the forearm, 0-1 mm and 30 mm (control site) from the incision. We defined categories from 0 (low) to 3 (high) to grade dilatation and elongation of the nutritive papillary capillaries, as well as the visibility of the superficial vascular plexus. Approximately 10 000 capillaries from 200 films were scored. RESULTS: The nutritive papillary capillaries were dilated and elongated (p < 0.01) after ≈24 h; that is, elongation (score 1.9 ± 0.9) vs baseline (score 0.9 ± 0.6), p < 0.01 and dilatation (score 2.2 ± 0.7) vs baseline (score 0.3 ± 0.3), p < 0.01. Superficial plexus visibility increased (p < 0.01) after ≈1 h (score 2.0 ± 0.7) and ≈24 h (score 2.7 ± 0.3) vs baseline (score 0.8 ± 0.4). CONCLUSION: The superficial vascular skin plexus showed enhanced visibility already ≈1 h after the skin trauma. Morphological remodeling in the nutritive papillary capillaries-dilatation and elongation after ≈24 h-facilitate increased O2  supply.

Effects of ischaemic postconditioning in aortic valve replacement: a multicenter randomized controlled trial.

OBJECTIVES: The effect of ischaemic postconditioning (IPost) on postcardioplegic cardiac function is not known. We hypothesized that IPost was cardioprotective in adult patients undergoing elective aortic valve replacement. METHODS: In a multicentre, prospective, randomized trial, patients (n = 209) were randomized to either a standard operation (controls) or postconditioning. Immediately before the cross-clamp was released, patients in the postconditioning group underwent 3 cycles of flow/non-flow (2 min each) of normothermic blood via the antegrade cardioplegia line. The primary end point was cardiac index. Secondary end points included additional haemodynamic measurements, biomarkers of cardiomyocyte injury, renal function parameters, intra- and postoperative arrhythmias and use of inotropic agents. RESULTS: There was no significant difference between the groups regarding cardiac index [mean between-group difference, 95% confidence interval (CI), 0.11 (-0.1 to 0.3), P = 0.27]. Postconditioning had no effect on other haemodynamic parametres. There was no between-group difference regarding troponin T or creatine kinase MB. Postconditioning reduced the relative risk for arrhythmias by 45% (P = 0.03) when postoperative atrial fibrillation and intraoperative ventricular fibrillation were combined. There were no differences in patients with/without diabetes, patients above/below 70 years of age or between the centres. However, after postconditioning, the cardiac index [95% CI, 0.46 (0.2-0.7), P = 0.001], cardiac output (P < 0.001), mean arterial pressure (P < 0.001) and left ventricular stroke work index (P < 0.001) were higher in males compared to females. CONCLUSIONS: IPost had no overall cardioprotective effects in patients undergoing aortic valve replacement but improved postoperative cardiac performance in men compared to women.

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.

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.

Survival predictor for penetrating cardiac injury; a 10-year consecutive cohort from a scandinavian trauma center.

BACKGROUND: Penetrating cardiac injuries in Europe have been poorly studied. We present a 10-year outcome for patients with penetrating heart injuries at Oslo University Hospital. METHODS: Data from 01.01.2001 until 31.12.2010 was collected from the Oslo University Hospital Trauma Registry and from the patients' records. RESULTS: Thirty-one patients were admitted with a penetrating cardiac injury. Fourteen patients survived (45%). Four out of 8 patients (50%) with gunshot wounds survived compared to 10 out of 23 (44%) with stab wounds. Median (quartiles) for the following values were: Injury Severity Score 25 (21-35), Revised Trauma Score 0 (0-6,9), Probability of Survival 0,015 (0,004-0,956), Glasgow Coma Scale 3 (3-13). Thirteen patients had signs of life on admission and survived. Eighteen patients were admitted without signs of life and received emergency department thoracotomy. Eight of these had no signs of life at the scene of injury and did not survive. Out of the remaining 10 patients, one survived. CONCLUSIONS: The outcome of patients with penetrating cardiac injury reaching the emergency department with signs of life was excellent. Hemodynamic instability indicates immediate surgery. Stable patients with penetrating thoracic trauma and possible cardiac injury detected by imaging should be considered for conservative treatment.

How to mend a broken heart: a major stab wound of the left ventricle.

A 28-year-old male admitted with a stab wound under his left nipple, underwent emergency surgery because of confusion, a decreasing blood pressure and increasing tachycardia. A median sternotomy incision was made and after establishing cardiopulmonary bypass, a 7 cm wound in the left ventricle and a smaller wound in the left atrium were repaired. An injured segment of lung was resected and the left anterior descending and circumflex arteries were grafted after weaning from cardiopulmonary bypass was initially unsuccessful. Although the patient suffered a stroke, probably due to prehospital hypoperfusion, he eventually recovered without major sequelae. In addition to the case report we present a literature review of the last 15 years pertaining the management of penetrating cardiac injury.

Postconditioning in mouse hearts is inhibited by blocking the reverse mode of the sodium-calcium exchanger.

Ischemic postconditioning and inhibition of the reverse mode of the sodium-calcium exchanger (NCX) are both cardioprotective. We hypothesized that a combination of these techniques might have an additive effect mediated by protein kinases (see below). Isolated perfused mouse hearts were subjected to 35 min of ischemia and 60 min of reperfusion. Each series had its own control ischemia group, the other groups were postconditioning with three cycles of 10 s of reperfusion and 10 s of ischemia immediately after sustained ischemia; the vehicle of the NCX blocker KB-R7943 was added to the perfusate 5 min before ischemia in series 1; KB-R7943 was added to the perfusate 5 min before ischemia with and without postconditioning in series 2; KB-R7943 was added to the perfusate for 5 min from the start of reperfusion with and without postconditioning in series 3. Infarct size was measured and cardiac function was evaluated. Phosphorylation of AKT, ERK1/2, PKCdelta and PKCepsilon was measured by immunoblotting. Postconditioning alone reduced infarct size by 37% and activated AKT (P=0.02). Blockade of NCX reduced infarct size when applied before ischemia (29%) and at start of reperfusion (32%). Combining NCX blockade with postconditioning abolished cardioprotection despite phosphorylation of ERK1/2 (P=0.03) and PKCepsilon (P=0.01).

Traumatic aortic valve rupture after a high-speed motor-vehicle accident: case report.

The case is reported of a 32-year-old male with an aortic valve injury received in a high-speed traffic accident. Several non-cardiac concomitant injuries were present, none of them life-threatening. Cardiac surgery was performed on day 2 due to a grade III aortic valve insufficiency. The presence of a large tear of the non-coronary cusp, and several concomitant injuries, led to valve replacement being considered the most optimal treatment for this patient.

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.

Traumatic aortic valve rupture after a high-speed motor vehicle accident.

The case is reported of a 32-year-old male who sustained an aortic valve injury after a high-speed traffic accident. Several non-cardiac concomitant injuries were present, none of which was life-threatening. Cardiac surgery was performed on day 2 due to a grade III aortic valve insufficiency. As a large tear of the non-coronary cusp was evident, together with several concomitant injuries, valve replacement was considered to be the most optimal treatment in this case.

Effects of hydrogen sulphide on ischaemia-reperfusion injury and ischaemic preconditioning in the isolated, perfused rat heart.

OBJECTIVE: Hydrogen sulphide (H(2)S) protects the heart against ischaemia-reperfusion injury caused by low flow or local ischaemia. We hypothesised that: (1) H(2)S protects against global ischaemia-reperfusion injury of the heart, (2) H(2)S plays a mechanistic role in ischaemic preconditioning, and (3) H(2)S acts by phosphorylation of protein kinases. METHODS: Isolated, perfused rat hearts were used in two series. Series 1: group 1.1 (n=10), 40 min of ischaemia and 120 min of reperfusion, group 1.2 (n=7), like 1.1 except that 40 microM NaHS was added to the perfusate during stabilisation and throughout the experiment. Group 1.3 (n=10), like 1.1, but endogenously produced H(2)S was blocked by D,L-propargylglycine. Series 2: group 2.1 (n=10) control, 30 min of ischaemia followed by 120 min of reperfusion. Group 2.2 (n=10) ischaemic preconditioning before sustained ischaemia and 120 min of reperfusion. Group 2.3 (n=10) like 2.2 except of D,L-propargylglycine treatment like in group 1.3. Mitogen activated protein kinases including extracellular signal-regulated kinases (ERK 1/2), the stress-activated/c-Jun NH2 terminal kinases (JNK), P38, as well as protein kinase B/AKT (AKT), adenosine monophosphate dependent protein kinase (AMPK) and the inducible heat shock protein 72 were measured by Western blotting. Adenine nucleotides (ATP, ADP, and AMP) were measured by high-pressure liquid chromatography and energy charge was calculated. RESULTS: Infarct size was increased by D,L-propargylglycine (40+/-6 vs 27+/-10% in controls, p=0.03, Bonferroni post hoc test). There was a non-significant decrease in infarct size in the NaHS group (to 20+/-13%). Western blot analysis indicated an upregulation of heat shock protein 72 in the NaHS treated group and a reduced phosphorylation of AKT in the D,L-propargylglycine group. D,L-propargylglycine had no effect on ischaemic preconditioning or on phosphorylation of protein kinases (ERK, AKT, P38, JNK and AMPK) in preconditioned hearts. No difference in energy charge was found between groups, although ADP was increased in the NaHS-treated group. CONCLUSION: Endogenous H(2)S production protects against global ischaemia, and H(2)S may be a part of the endogenous cell defence. However, endogenous H(2)S did not appear to be important in ischaemic preconditioning, and protein kinases were not important for the effect of H(2)S. Exogenous H(2)S may provide myocardial protection, possibly acting by expression of heat shock protein 72.

Preconditioning effects of steroids and hyperoxia on cardiac ischemia-reperfusion injury and vascular reactivity.

OBJECTIVE: Corticosteroids and hyperoxia protect the heart against ischemia-reperfusion injury and may attenuate vascular reactivity. We hypothesized that (1) combining these two pretreatments induces an additive cardioprotection, (2) protection depends on activation of survival kinases and/or heat shock proteins, and (3) these interventions would change vascular reactivity into a more relaxed state. METHODS: Male rats were randomized (n=10 in each group): 1. control, 2. dexamethasone (3mg/kg) injected 24 and 12 h before harvesting the hearts, 3. 60 min of hyperoxia (90-95% O(2)) immediately before harvest, 4. combination of dexamethasone and hyperoxia as in groups 2 and 3. The hearts were Langendorff-perfused and exposed to 30 min of global ischemia and reperfused for 120 min. Cardiac function was monitored and infarct size determined. Isometric tension to vasoconstrictive and vasodilatory agents was measured in femoral artery rings. Phosphorylation of survival kinases (protein kinase B/AKT, extracellular signal-regulated kinases (ERK1/2), the stress-activated/c-Jun NH2 terminal kinases (SAPK/JNK) and p38 MAPK), adenosine monophosphate dependent kinase (AMPK) and expression of heat shock protein 72 (HSP72) in hearts was evaluated by immunoblotting. RESULTS: Infarct size was attenuated in all pretreated groups versus controls: 29% reduction in the combined group (p<0.01), 23% in hyperoxia group (p<0.05) and 31% in dexamethasone group (p<0.01). There was no significant difference between the treated groups. Combined pretreatment improved postischemic left ventricular end diastolic pressure compared to all other groups (p<0.001 vs controls, p=0.002 vs dexamethasone, p=0.005 vs hyperoxia). Combined pretreatment improved left ventricular developed pressure and coronary flow compared to controls (p<0.001 for both) and hyperoxia (p=0.0047 and p=0.0024, respectively). Combined pretreatment enhanced endothelium-independent relaxation (p=0.0032) compared to controls. Excepting ERK1/2 phosphorylation in the combined group during early reperfusion, there was no increased phosphorylation of the survival kinases AKT, p38, JNK, or AMPK and no increase of HSP72 expression. CONCLUSION: Combined pretreatment by hyperoxia and dexamethasone improved postischemic heart function, but did not reduce infarct size compared to single pretreatment groups. Except of a possible role of ERK1/2, protection depended neither on survival kinases nor heat shock protein 72.

Postconditioning in rats and mice.

OBJECTIVE: For subsequent studies on the molecular mechanisms of postconditioning, we aimed to identify a robust postconditioning protocol in rat and mouse heart. DESIGN: Isolated rat hearts were subjected to different postconditioning protocols (study 1 and 2). The protection was compared to preconditioning. Rats (study 3) in vivo in two different laboratories were postconditioned. Isolated mouse hearts (study 4) and mice in vivo (study 5) were postconditioned. RESULTS: Postconditioning did not protect isolated, perfused rat hearts, however, preconditioning improved function and reduced infarct size. Postconditioning tended to protect rat hearts in vivo in one laboratory (p = 0.10), whereas protection was seen in the other laboratory (infarct size 51+/-11% vs controls 62+/-3%, p = 0.01). Postconditioned mouse hearts were protected, both ex vivo (16+/-9% vs controls 33+/-18%, p = 0.02) and in vivo (21+/-5% vs 42+/-7%, p < 0.001). CONCLUSIONS: Rat hearts are less suitable for studies of mechanisms of postconditioning. The results suggest that the signaling pathways differ between pre- and postconditioning. Mouse hearts were strongly protected by postconditioning, and genetically engineered mice may be useful for postconditioning research.

Cardiac tamponade caused by acute spontaneous coronary artery rupture.

Acute spontaneous coronary artery rupture is rare and the diagnosis might be missed due to high risk of mors subita. We present three patients hospitalized with signs of cardiac tamponade due to acute spontaneous coronary artery rupture. All the three were successfully operated with evacuation of the pericardial hematoma, identification of the bleeding site, and hemostasis. The patients were examined with coronary angiography and computer tomography, and no underlying cause of the rupture was detected. In patients presenting with cardiac tamponade, acute spontaneous coronary artery rupture is a possible diagnosis.